Unraveling of interacting protein network of chaperonin TCP1 gamma subunit of Leishmania donovani

Abstract

T-complex polypeptide-1 (TCP1) is a group II chaperonin that folds various cellular proteins. About 10% of cytosolic proteins in yeast have been shown to flux through the TCP1 protein complex indicating that it interacts and folds a plethora of substrate proteins that perform essential functions. In Leishmania donovani, the gamma subunit of TCP1 (LdTCP1γ) has been shown to form a homo-oligomeric complex and exhibited ATP-dependent protein folding activity. LdTCP1γ is essential for the growth and infectivity of the parasite. The interacting partners of L. donovani TCP1γ, involved in many cellular processes, are far from being understood. In this study, we utilized co-immunoprecipitation assay coupled with liquid chromatography–mass spectrometry (LC–MS) to unravel protein–protein interaction (PPI) networks of LdTCP1γ in the L. donovani parasite. Label-free quantification (LFQ) proteomic analysis revealed 719 interacting partners of LdTCP1γ. String analysis showed that LdTCP1γ interacts with all subunits of TCP1 complex as well as other proteins belonging to pathways like metabolic process, ribosome, protein folding, sorting, and degradation. Trypanothione reductase, identified as one of the interacting partners, is refolded by LdTCP1γ. In addition, the differential expression of LdTCP1γ modulates the trypanothione reductase activity in L. donovani parasite. The study provides novel insight into the role of LdTCP1γ that will pave the way to better understand parasite biology by identifying the interacting partners of this chaperonin.

Supplementary Information

The online version contains supplementary material available at 10.1007/s12192-022-01262-4.

Introduction

Leishmaniasis is caused by the obligate protozoan parasite of the genus Leishmania (Torres-Guerrero et al. 2017). The disease is clinically characterized into four forms: cutaneous leishmaniasis (CL), mucocutaneous leishmaniasis (MCL), visceral leishmaniasis (VL), and post kala-azar dermal leishmaniasis (PKDL) (WHO 2020). VL is fatal if left untreated and causes 20,000–30,000 deaths annually (https://www.dndi.org/diseases-projects/leishmaniasis/). According to the WHO report, more than 95% of VL cases occur in 10 countries: India, Nepal, China, Iraq, Kenya, Somalia, Brazil, Ethiopia, South Sudan, and Sudan (WHO 2020).

Leishmania spp. leads a digenetic life cycle in two distinct morphological forms: promastigote in sand fly vector and amastigote in macrophages of the mammalian host (Molyneux and Killick-Kendrick 1987). Leishmania promastigotes differentiate to intracellular amastigotes in the distinct environment of the phagolysosome of the mammalian host (Tsigankov et al. 2014). During this differentiation process, Leishmania parasites are exposed to a variety of environmental stress such as temperature, pH, oxidative stress, and nutritional deprivation, which can damage the cellular structure and interfere with critical parasite functions (Garlapati et al. 1999; Degrossoli et al. 2004; Anas et al. 2019). Heat shock proteins (HSPs), a subset of molecular chaperones, are known to counteract these stresses by folding, assembling, secreting, and regulating various proteins, and hence play a crucial role in parasite survival (Degrossoli et al. 2004; Hombach et al. 2014). Several of these HSPs are also involved in the maintenance of the structural integrity of cellular proteins under normal physiological conditions (Saibil 2013). HSPs are subdivided into different families: small HSPs, HSP40, HSP60/chaperonins, HSP70, HSP83/90, HSP100, and HSP110 based on their molecular masses (Smith et al. 1998; Feder and Hofmann 1999). HSPs are also known to be involved in protein trafficking, cell adhesion, drug resistance, modulation of host immune response, and export of virulence factors in many of the intracellular protozoan parasites (Anas et al. 2019). Leishmania HSP100, shown to be essential for the survival of the amastigote stage of the parasite, plays a critical role in exosome biogenesis and host immune modulation (Krobitsch and Clos 1999; Hubel et al. 1997; Silverman et al. 2010b). Further, HSP83/90 has been found to be involved in the proliferation and cell cycle of the Leishmania parasite (Hombach and Clos 2014).

TRiC (TCP1 ring complex) or TCP-1 (T-complex polypeptide-1) or CCT (chaperonin-containing TCP1) is a group II chaperonin, forms a high molecular weight hetero-oligomeric protein complex with two identical rings in archaea and in the cytosol of eukaryotes (Spiess et al. 2004; Hartl et al. 2011). TCP1 rings include eight–nine paralogous subunits that mediate ATP-dependent folding and assembly of a wide range of cytosolic proteins (Kubota et al. 1995). Functional roles of monomeric, homo-oligomeric, and complexes with two or more TCP1 subunits having independent activities have been demonstrated in eukaryotes (Echbarthi et al. 2018; Brackley and Grantham 2010). We previously studied L. donovani TCP1γ subunit (LdTCP1γ) and showed that it is organized into double toroidal rings of seven subunits, forming a homo-oligomeric complex in L. donovani and also exhibited ATP-dependent refolding activity (Bhaskar et al. 2015). The inability of LdTCP1γ double-allele replacement mutants to survive clearly established its essentiality for parasite survival (Yadav et al. 2020b). LdTCP1γ is required for the growth and infectivity of the parasite and can be employed as a drug target (Yadav et al. 2020b). Further, LdTCP1γ has been shown to protect the parasite from miltefosine-induced oxidative stress (Yadav et al. 2020a).

In the present study, we, for the first time deduced the protein interaction network of LdTCP1γ in L. donovani parasite. The interacting partners of LdTCP1γ were pulled out by co-IP (co-immunoprecipitation) followed by protein identification by liquid chromatography with tandem mass spectrometry (LC–MS/MS) and western blot analysis. Total 719 proteins were enriched with a fold change greater than 4.5 in the co-IP samples using anti-LdTCP1γ polyclonal antibodies as compared with control samples done with pre-immune serum. The identified proteins were represented by various pathways like metabolic process, ribosome, proteasome, protein folding, sorting, and degradation. One of these identified proteins, trypanothione reductase, a key enzyme in the thiol redox homeostasis pathway (Mittal et al. 2005), was chosen for further studies. LdTCP1γ was also able to fold denatured trypanothione reductase into its native state. Thus, the present study provides new insights into the novel functions of LdTCP1γ and helps to understand parasite biology.

Methods

Parasite and cell culture

L. donovani Dd8 promastigotes (MHOM/IN/80/Dd8; World Health Organization reference strain of Indian origin) were originally obtained from the late Prof. P.C.C. Garnham (Imperial College, London, UK). In the present study, this laboratory/wild-type strain was designated as Dd8^+/+. The parasite was regularly passaged in golden hamsters at the CSIR-Central Drug Research Institute to maintain its virulence. Dd8^+/+ parasites were grown in medium 199 (Sigma, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco, Waltham, MA, USA), 1% antibiotic–antimycotic solution (Sigma) at 24 °C (Debrabant et al. 1995).

LdTCP1γ overexpression construct was obtained by cloning LdTCP1γ gene in pKS-neo shuttle vector, and the construct was transfected into wild-type promastigotes (Dd8^+/+) to obtain LdTCP1γ overexpression transfectants (Dd8^++/++) as described previously (Yadav et al. 2020b). LdTCP1γ overexpression transfectants (Dd8^++/++, exhibiting increased LdTCP1γ expression compared with wild-type parasite) or the vector control (Dd8^Vc; LdTCP1γ expression similar to wild-type parasite), were selected and maintained in the presence of G418 (20 µg/ml) in M199 medium supplemented with 10% FBS (Yadav et al. 2020b).

Targeted gene replacement using homologous recombination was performed to replace both alleles of LdTCP1γ with hygromycin and neomycin phosphotransferase genes to generate single-allele replacement and double-allele replacement mutants, respectively (Yadav et al. 2020b). The respective transfectants were selected and maintained in 40 µg/ml hygromycin and 20 µg/ml G418 in M199 medium supplemented with 10% FBS. Since double-allele replacement mutants failed to survive, single-allele replacement mutants (Dd8^+/-, exhibiting decreased LdTCP1γ protein expression compared with wild-type parasite) were used for further experiments.

Episomal complementation mutants (Dd8^+/-/+) were developed by transfecting LdTCP1γ overexpression construct into the single-allele replacement parasite as described previously (Yadav et al. 2020b). Dd8^+/-/+ mutant parasites (exhibiting protein expression similar to wild-type parasites) were maintained under 40 µg/ml hygromycin and 20 µg/ml G418 drug pressure in M199 medium supplemented with 10% (v/v) FBS.

Protein expression and purification

L. donovani trypanothione reductase was expressed in E. coli BL21 (DE3) and purified as described earlier (Mittal et al. 2005). LdTCP1γ was expressed in E. coli C41 (DE3) and purified as described previously (Bhaskar et al. 2012). The purity of both the recombinant proteins was checked on 10% SDS-PAGE as described previously (Laemmli 1970). The Bradford method was used to quantify proteins using bovine serum albumin (BSA) as the standard (Bradford 1976).

Antibody production

Purified recombinant LdTCP1γ protein and synthetic peptides of LdTCP1ε (EKKEFLLQTARI) or LdTCP1δ (ERKYLLGLCKAIKDA) (GL Biochem, China) were used for immunization. 1-ethyl-3-(3 dimethylaminopropyl)-carbodiimide (EDC) (Thermo Scientific) was used as a coupling agent to crosslink Keyhole limpet hemocyanin (KLH) with peptides using the manufacturer’s protocol. Polyclonal antibodies were raised in NZW rabbits (6 weeks old) against recombinant LdTCP1γ protein or LdTCP1ε/ LdTCP1δ KLH conjugated peptides. Pre-immune sera were collected from each rabbit prior to immunization. The emulsion of 100 µg purified protein/ synthetic peptide with an equal volume of complete Freund’s adjuvant (Sigma) was prepared and injected subcutaneously. After 2-week intervals, two boosters of 100 µg protein emulsified with incomplete Freund’s adjuvant were injected. The antiserum was collected after 10 days of the last immunization and stored at – 20 °C. The polyclonal antiserum was purified, as reported earlier (Bajaj et al. 2020).

Co-immunoprecipitation assay

A co-immunoprecipitation assay was performed as described previously (Bhaskar et al. 2015). Briefly, log-phase promastigotes (3 × 10⁸) of L. donovani were washed three times with 1 × PBS and sonicated in 1 ml lysis buffer (15 mM HEPES, pH 7.4; 150 mM NaCl; 10 mM MgCl₂; 5 mM EGTA; 2 mM EDTA; 0.5% v/v Nonidet P-40; 1 mM phenylmethanesulfonyl fluoride (all chemicals from Sigma)). The lysate was centrifuged at 16,000 × g for 30 min at 4 °C. The supernatant was taken as cell lysate. ‘Cell lysate control’ was prepared by mixing 20 µl of cell lysate with 5 × SDS-sample buffer (312.5 mM Tris–HCl (pH 6.8), 50% glycerol, 10% SDS, 10% β-mercaptoethanol, 0.25% bromophenol blue). The rest cell lysate was mixed with 50 µl Dynabeads Protein G (Thermo Fischer Scientific) that had been conjugated to rabbit pre-immune (PI) sera or LdTCP1γ polyclonal antibodies (1:100, raised in rabbit) using an irreversible crosslinker BS3 (Thermo Fischer Scientific) for 30 min at room temperature. The mixture was neutralized with 50 mM Tris (pH 7.4) and incubated for 3 h at 4 °C. After washing beads with lysis buffer, bound proteins were eluted in SDT buffer (2% SDS; 100 mM DTT; 150 mM Tris–HCl, pH 8.0) or 1 × SDS sample buffer by incubating for 30 min at 37 °C in a water bath. A magnetic rack (Life Technologies) was used to separate the supernatant from beads. The samples were subjected to LC–MS/MS analysis or loaded on 10% SDS-PAGE followed by western blotting.

Western blotting

Proteins were separated on 10% SDS-PAGE, transferred onto PVDF membrane, and subjected to western blot analysis as described previously (Laemmli 1970; Towbin et al. 1979). The membrane was probed with rabbit polyclonal anti-LdTCP1γ (1:5000) (Bhaskar et al. 2012) or chicken polyclonal anti-HSP90 (1:3000) (Brandau et al. 1995) or chicken polyclonal anti-HSP70 (1:3000) (Brandau et al. 1995) or rabbit polyclonal anti-TR (1:3000) or rabbit polyclonal anti-TryPx (1:3000) (Singh et al. 2017) or mouse monoclonal anti-α-Tubulin (Sigma, 1:20,000) antibodies. HRP conjugated goat anti-rabbit IgG (GE Healthcare, 1:10,000) or HRP conjugated goat anti-chicken (Santa Cruz, 1:10,000) or HRP conjugated goat anti-mouse IgG (GE Healthcare, 1:10,000) were used as secondary antibodies. The ECL reagent (GE Healthcare) was used to develop blot as per the manufacturer’s instructions. The blot was visualized on MyECL chemiluminescent Imager (Thermo Fischer Scientific). ImageJ (version 1.51) (Rueden et al. 2017) software was used for densitometric analysis of the immunoblots.

Sample preparation for nano-LC–MS/MS

The sample was prepared using the filter-aided sample preparation (FASP) method as described previously (Zhu et al. 2016). After elution of bound proteins in 30 µl SDT buffer, DTT, and other low-molecular-weight components were removed and replaced with 200 μl UA buffer (8 M Urea; 150 mM Tris–HCl, pH 8.0) by repeated ultrafiltration using amicon filter (Millipore, Amicon Ultra 0.5 ml filter, 10 kDa). Subsequently, the samples were carboxymethylated with iodoacetamide (IAA, 100 µM in UA) in the dark for 30 min at room temperature. The samples were then centrifuged for 10 min at 14,000 × g. Amicon filters were then washed three times with 100 μl UA buffer and twice with 100 μl of ABC buffer (50 mM ammonium bicarbonate, pH 8.5). Samples were adjusted to 50 μl with 50 mM ABC buffer and incubated with 50 µl of 1 µg Trypsin (Trypsin singles, Sigma) overnight at 37 °C in the amicon filters. The digested samples were centrifuged for 30 min at 14,000 × g, and the filtrate was collected. The filtrates containing digested peptides were dried completely using a vacuum centrifuge (Christ). The peptides were desalted using C18 ZipTip (Millipore) as per manufacturer’s instruction and eluted in 10 µl elution buffer (0.1% formic acid in 60% acetonitrile).

Nano-LC MS/MS (nano-liquid chromatography–tandem mass spectrometry) analysis

The label-free quantification analysis of the trypsin digested peptides was performed using Q Exactive Plus mass spectrometer (Thermo Fischer Scientific) integrated with EASY-nLC™ 1200 System (Thermo Fischer Scientific) and a nanoelectrospray ion source (Thermo Fischer Scientific) (ILBS; Institute of liver and biliary sciences, New Delhi, India) as described previously (Kaur et al. 2021). Peptides were separated by using a 50-cm Proteo-Prep C18 column having an inner diameter of 75 µm. A buffer containing 0.1% formic acid was used to load peptide samples and a buffer containing 80% acetonitrile and 0.1% formic acid (v/v) was used to elute peptides with a 150-min linear gradient, at a flow rate of 300 nl/min. The data-dependent top 20 system was utilized to obtain mass spectra, with an automatic changeover between MS and MS/MS. To collect mass spectra, the Orbitrap analyzer was utilized with a mass range of 300–1800 m/z and 70,000 resolutions at 200 m/z. The HCD peptide fragments were obtained using a normalized collision energy of 27 kcal/mol. Survey scan and MS/MS scan maximal ion injection periods were 20 and 100 ms correspondingly with 3e6 and 2e5 as ion target values. Xcalibur software was used to acquire data.

Data analysis

The mass spectrometry data were analyzed in Proteome-Discoverer software (Thermo-Scientific, V2.3) using the label-free quantitation algorithm for quantitation of the precursor ion, as described previously (Kaur et al. 2021). Mascot and Sequest databases were simultaneously utilized for searching the MS/MS spectra against the UniProt L. donovani protein databases (UP000008980 and UP000274082). Data analysis settings included carbamidomethylation (C) as a fixed modification, trypsin as protease (one missed cleavage was allowed), and methionine oxidation as a variable modification. The mass error tolerances for the precursor and product ions were adjusted to 20 ppm and 0.1 Da, respectively. Peptides having confidence scores of > 0.05 were evaluated for further analysis, and bias correction was applied automatically. Peptide and product ion tolerance of 0.05 Da was utilized for the searches. Protein identification was allowed with a local false discovery rate of less than 1% and a minimum of one unique peptide with a threshold confidence of 95%. The supplementary material contains all the information related to the identified peptides and proteins in this study.

Bioinformatic analysis

Downstream statistical analysis of data was performed using open-source software and MetaboAnalyst 5.0 (Chong et al. 2019) open-source web-based tool using standard parameters. The normalized abundances of identified proteins obtained from the Proteome-Discoverer software (Thermo-Scientific, V2.3), was used to perform all the downstream statistical analyses. MetaboAnalyst 5.0 was used to generate Principal Component Analysis (PCA) and volcano plot. Microsoft Excel 2010 was used to perform two-sample t tests to produce functional, statistical comparisons of the groups. Gene ontology analysis was performed using Funrich software (version 3.1.4) (Pathan et al. 2015), including only those ontological terms that are associated with the protein members. Protein network analysis was performed using stringApp of Cytoscape software (version 3.8.2) (Shannon et al. 2003).

Chemical denaturation, refolding, and enzyme activity of recombinant trypanothione reductase

Recombinant purified trypanothione reductase (TR) was chemically denatured using guanidinium hydrochloride (GdmCl) as described earlier (Rai et al. 2009). Briefly, 4.4 nM TR was incubated with 2.5 M GdmCl for 1 h at 25 °C in denaturing buffer containing 25 mM HEPES (pH 7.4) and 50 mM potassium acetate. Then denatured TR was refolded in buffer containing 25 mM HEPES (pH 7.4), 100 mM potassium acetate, 10 mM magnesium acetate, 1 mM ATP with or without 0.5–1 µM recombinant LdTCP1γ (based on molarity, calculated as a tetradecamer subunit complex). TR enzyme activity was measured spectrophotometrically at 412 nm as described previously (Hamilton et al. 2003).

Trypanothione reductase activity assay in crude cell lysate

Assessment of trypanothione reductase activity was performed in the cell lysate of L. donovani promastigotes. After washing with 1X PBS, cells were lysed for 15 min in a lysis buffer containing 1 mM EDTA, 40 mM HEPES, 2% Triton X-100, and 1 mM phenylmethanesulfonyl fluoride (PMSF) (all chemicals from Sigma) (van den Bogaart et al. 2014). Trypanothione reductase activity in cell lysates of Dd8^+/+, Dd8^Vc, Dd8^+/−, Dd8^++/++, and Dd8^+/-/+ cell lines was assessed spectrophotometrically at 412 nm, as described previously (Hamilton et al. 2003).

Statistical analysis

The data were expressed as means ± standard error unless otherwise stated. Comparisons were made using one-way analysis of variance (ANOVA) by GraphPad Prism software version 5 (GraphPad Prism Software, San Diego, CA, USA), and differences at a P < 0.001 (***) or < 0.01 (**) or < 0.05 (*) were considered significant; a P > 0.05 was considered nonsignificant (ns).

Results

Identification of LdTCP1γ interacting proteins in L. donovani parasite using co-IP and LC–MS/MS

Six independent co-immunoprecipitation (co-IP) assays were performed, three employing LdTCP1γ antibodies and three with pre-immune sera (control). A total of 1361 proteins were identified from LC–MS/MS–based label-free quantification proteomics analysis. However, 121 proteins with a single value across the sample were removed to obtain 1240 proteins (Table S1). Principle component analysis (PCA) using the partial least squares–discriminant analysis (PLS-DA) model was performed to obtain a global picture of the variations between samples (Fig. 1a). Results showed that both samples clustered separately along with the 1st component, indicating statistical variance among them (Fig. 1a). The biological replicates of both samples were also clustered together representing their reproducibility (Fig. 1a). Out of 1240 proteins, 719 proteins were significantly enriched (P < 0.05) with greater than 4.5-fold change compared with the control group (Table S1 and Fig. 1b).

Fig. 1.

Analysis of LdTCP1γ interacting proteins by LC–MS/MS. a Principal component analysis (PCA) plot using partial least squares discriminant analysis (PLS-DA) model showing the inter-sample correlation between co-IP sample using LdTCP1γ antibody (LdTCP1γ-co-IP) and co-IP sample using pre-immune sera (control). b Volcano plot of identified proteins (1240 proteins) showing proteins with significant fold change in LdTCP1γ co-IP samples compared with control samples. Proteins with > 4.5-fold change are indicated with pink color, while remaining proteins are shown with gray color

Functional annotation and pathway enrichment analysis of LdTCP1γ interacting partners

To further investigate the functional annotation of 719 proteins, GO enrichment analyses were performed based on biological processes and molecular functions using FunRich software. The significantly enriched (P < 0.05) biological processes GO terms comprised of translation, protein folding, tricarboxylic acid cycle, nucleotide biosynthetic process, nucleoside metabolic process, modulation by symbiont of host immune response, symbiont defense to host-produced nitric oxide, regulation of protein catabolic process, formation of cytoplasmic translation initiation complex, glycolytic process, fatty acid beta-oxidation, peroxisome fission, and suppression by symbiont of host immune response (Fig. 2a), while highly significant enriched (P < 0.001) molecular function GO terms were structural constituent of ribosome, translation initiation factor activity, proton-transporting ATP synthase activity, unfolded protein binding, enzyme regulator activity, and ribose phosphate diphosphokinase activity (Fig. 2b). Significantly enriched (P < 0.05) KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways include ribosome, metabolic pathways, protein folding/sorting, and proteasome (Fig. 2c).

Fig. 2.

The GO term enrichment, associated with the interacting partners of LdTCP1γ. a Biological process. b Molecular function. c KEGG pathway analysis

Generation of protein–protein interaction (PPI) network based on identified LdTCP1γ interacting proteins

To confirm the interactions detected experimentally, the STRING database was used, which manages diverse sources of known and predicted PPIs, facilitating a comparative analysis (Szklarczyk et al. 2019). Both physical and functional protein associations were considered to create a PPI network. The LdTCP1γ interacting proteins in the PPI network were categorized and clustered together using string enrichment application of Cytoscape software based on KEGG pathway analysis (Fig. 3). LdTCP1γ is represented by the yellow node, while interaction partners are represented by the remaining nodes (Fig. 3). The direct interacting partners of LdTCP1γ (Uniprot Id: E9BGG0) are represented by red nodes. These include all seven subunits of TCP1 complex namely TCP1α (E9BP97), TCP1β (E9BJ47), TCP1δ (E9BFB9), TCP1ε (E9BNK9), TCP1ζ (E9BBL4), TCP1η (E9BSR0), and TCP1θ (E9BV80); putative heat shock protein (hsp70 family); heat shock protein 10; clathrin heavy chain; elongation factor-1 gamma; eukaryotic translation release factor and translation initiation factor EIF-2B gamma; and guanine nucleotide-binding subunit beta-like protein (Fig. 3 and Table S1). Other directly interacting partners (red nodes) were categorized in the enriched pathways obtained from the string enrichment analysis. These include putative 40S ribosomal protein S3, ribosomal protein L1a, ribosomal protein L3, 40S ribosomal protein S4 and 60S ribosomal protein L10 from the ribosome pathway; putative proteasome regulatory ATPase subunit 1 and subunit 5 from the proteasome pathway; ATPase alpha subunit, ATP synthase beta subunit, and aldolase from the metabolic pathway; and heat shock protein 83/90 from protein folding and sorting pathway (Tables 1, 2, 3, and S1). Several proteins that lack any clear orthologues to other organisms and significantly interacted with LdTCPγ (14–43-fold change) were classified as ‘uncharacterized proteins’ (Table 4).

Fig. 3.

Protein–protein interaction network analysis of LdTCP1γ interacting partners using stringApp of Cytoscape software. Proteins are shown in clusters that are associated with the particular KEGG pathway. LdTCP1γ is shown with the yellow hexagonal box. Proteins from the metabolic pathway shown with pink box, ribosome pathway associated proteins with purple box, proteasome pathway associated proteins with green box, and proteins from folding, sorting, and degradation pathway are indicated with the blue box. The direct interacting proteins of LdTCP1γ are shown with the red box

Table 1.

List of interacting proteins involved in metabolic pathway (map01100)

Accession	Description	MW (kDa)	Coverage (%)	# Unique peptides	Fold change (TCP1γ /PI)	P value
Fatty acid metabolism
E9BQL7	Putative enoyl-[acyl-carrier-protein] reductase	33.9	9	2	15.25	0.0027
E9BH95	Putative fatty-acid desaturase	49	7	3	6.43	0.0002
A0A3S5H7Q0	3-Ketoacyl-CoA thiolase-like protein, putative	47	12	6	5.84	0.0064
E9BN78	Acetyl-CoA carboxylase	241	2	4	11.38	0.0445
Glycolysis/gluconeogenesis/TCA cycle
E9BC06	Enolase	46	23	7	29.52	0.0157
E9BG61	Putative NADP-dependent alcohol dehydrogenase	38.4	9	3	46.31	0.0134
E9BM86	Phosphoglycerate kinase	57.6	5	3	15.96	0.0038
E9BTJ1	Fructose-bisphosphate aldolase	40.7	16	5	16.62	0.0006
E9BM42	Glyceraldehyde-3-phosphate dehydrogenase	39.1	27	10	5.25	0.0092
E9BPA1	Dihydrolipoyl dehydrogenase	50.6	14	5	7.12	0.0458
A0A3Q8IBX3	Phosphotransferase	51.7	10	4	7.36	0.0022
E9BHT3	Pyruvate dehydrogenase E1 component subunit beta	37.9	32	9	4.65	0.0255
E9BKZ2	Dihydrolipoamide dehydrogenase, putative	56.3	11	5	40.12	0.0026
E9BA79	Isocitrate dehydrogenase (NADP)	48.5	20	8	13.10	0.0008
E9BK25	Malate dehydrogenase	34.1	15	4	14.84	0.0077
Oxidative phosphorylation
E9BIW4	Acyl carrier protein	16.7	6	1	13.48	0.0014
E9BFJ4	Putative cytochrome c oxidase subunit VI	19.2	16	3	8.20	0.0149
E9BFK0	Putative ATP synthase F1 subunit gamma	34.4	9	2	25.51	0.0111
E9BMA9	Putative ATP synthase, epsilon chain	20.1	4	1	8.78	0.0003
E9B8E1	ATPase alpha subunit	62.5	11	6	5.20	0.0099
A0A3S5H5L6	Putative ATPase	55.2	6	2	4.63	0.0180
E9BHM5	ATP synthase subunit beta	56.3	21	8	19.14	0.0016
E9BG58	V-type proton ATPase subunit G	12.4	15	1	8.72	0.0012
E9BH32	Succinate dehydrogenase	66.7	5	2	5.40	0.0020
E9BN35	Ubiquinol-cytochrome-c reductase-like protein	7.9	13	1	6.54	0.0157
A0A3S7WV61	Plasma membrane ATPase	107.3	7	6	10.32	0.0021
Pentose phosphate pathway
E9B9F7	Putative ribose-phosphate pyrophosphokinase	93.7	9	7	10.74	0.0006
E9BUS0	Putative phosphoribosylpyrophosphate synthetase	39.3	18	6	11.33	0.0000
E9BRF6	Phosphomannomutase-like protein	65.2	3	1	5.13	0.0363
Glutathione metabolism
E9B8C5	Trypanothione reductase	53	2	1	9.77	0.0037
E9BG25	Putative peroxidoxin	25.4	25	6	4.58	0.0027
E9BCF2	Tryparedoxin peroxidase	22.2	37	7	7.87	0.0024
E9BI90	Putative glutathione peroxidase	19.4	19	9	54.26	0.0058
Glycerolipid metabolism
A0A3S7WPJ1	Putative lipin	142.6	2	2	12.57	0.0004
E9BMN9	Putative monoglyceride lipase	34.6	4	1	4.80	0.0189
E9BSI3	Glycerol kinase	55.9	11	5	15.21	0.0009
Biosynthesis of amino acids
E9BKW6	Putative glutamamyl carboxypeptidase	44	10	3	13.76	0.0253
E9BBL6	Pyrroline-5-carboxylate reductase	28.6	6	1	4.79	0.0166
A0A3Q8IGG4	Branched-chain amino acid aminotransferase, putative	44.1	2	1	5.22	0.0208
A0A3Q8IG44	S-adenosyl-methionine synthase	43.1	14	5	5.70	0.0005
E9BMC4	5-Methyl tetrahydropteroyl triglutamate	85.9	12	10	9.52	0.0018
E9BJ27	Cysteine desulfurase	47.8	2	1	7.36	0.0037
E9BIH1	Proline dehydrogenase	63.7	5	3	12.01	0.0040
E9BS25	Arginase	36.1	5	2	13.10	0.0013
A0A3S7WTK6	NAD-specific glutamate dehydrogenase	115	5	5	18.41	0.0066
Purine metabolism
E9BFD5	Putative adenylate kinase	30	9	2	12.22	0.0099
E9B9K4	Phosphoribosylpyrophosphate synthetase	40.8	3	1	7.55	0.0270
E9BC44	ATP diphosphohydrolase	47.2	4	1	6.84	0.0005
E9BP61	Nucleoside diphosphate kinase	16.6	26	3	52.54	0.0064
Steroid biosynthesis
E9BQ02	Sterol C-24 reductase, putative	58.1	3	1	4.77	0.0137
E9BBL0	Squalene monooxygenase	63.5	15	7	13.59	0.0012
A0A3Q8IK91	Methyltransferase	39.9	2	1	12.38	0.0134
Miscellaneous
E9BLA7	Alkyldihydroxyacetonephosphate synthase	69.4	29	19	7.89	0.0008
E9BAP8	3-Methylcrotonoyl-CoA carboxylase beta subunit	58.4	10	5	6.98	0.0047
E9BJ13	Putative isovaleryl-coA dehydrogenase	44.4	3	1	13.71	0.0005
A0A3Q8IDH5	D-lactate dehydrogenase-like protein	53.7	7	4	28.10	0.0009
E9BE31	Lipoyl synthase, mitochondrial	45.1	1	1	9.60	0.0100
A0A3S7XBH2	Amino methyl -transferase	41.5	3	1	6.40	0.0114
E9BSH7	1-Alkyl-2- acetylglycero phosphocholine esterase	49.8	8	3	22.55	0.0070
E9BUB8	Adenosylhomocysteinase	47.7	11	5	5.50	0.0293
A0A3Q8IEE8	GDP-mannose pyrophosphorylase	41.7	11	4	6.43	0.0005
A0A3S7X8D9	Serine palmitoyltransferase-like protein	53.9	2	1	5.19	0.0007
E9BUH3	Asparaginase	41.2	25	9	6.70	0.0272
A0A3Q8IFK1	Adenine phosphoribosyltransferase	26.2	9	2	11.36	0.0030
A0A3S7X4X4	Putative 2-oxoglutarate dehydrogenase	67.5	11	6	24.87	0.0196
E9BE90	4-Coumarate: coa ligase -like protein	66.1	36	15	9.38	0.0002

Table 2.

List of interacting proteins belonging to the category ‘Ribosome’ (map03010)

Accession	Protein name	MW (kDa)	Coverage (%)	# Unique peptides	Fold change (TCP1γ /PI)	P value
Ribosome
A0A3S7X0P4	60S acidic ribosomal protein P0	34.7	19	4	16.21	0.0384
E9BS76	60S ribosomal protein L32	15.4	6	1	15.66	0.0041
A0A3S7WWY7	40S ribosomal protein S6	28.3	16	3	10.47	0.0189
E9BHM6	40S ribosomal protein S25	13	18	2	10.47	0.0430
E9BMC1	Putative 60S acidic ribosomal protein P2	10.5	55	4	7.76	0.0071
E9B8R9	Putative 60S ribosomal protein L23a	16.3	37	5	19.00	0.0345
E9BBC0	Putative 40S ribosomal protein S12	15.6	22	3	25.19	0.0071
E9BF65	Putative 60S ribosomal protein L36	12	6	1	6.58	0.0368
E9BGL6	Putative 60S ribosomal protein L17	19.1	26	4	7.74	0.0004
A0A3Q8IED5	40S ribosomal protein S2	28.7	12	3	5.88	0.0330
A0A3Q8IGG0	Putative ribosomal protein L3	47.6	14	5	13.12	0.0300
A0A3Q8IAA6	Putative 60S ribosomal protein L6	21.2	4	1	5.40	0.0060
A0A3Q8INI0	Putative 40S ribosomal protein S19 protein	20.2	20	1	22.92	0.0087
E9BRQ6	60S ribosomal protein L30	11.4	35	3	42.75	0.0354
E9BRS2	Putative 40S ribosomal protein S3A	30	20	5	27.22	0.0082
E9BSQ4	Putative 60S ribosomal protein L23	14.9	50	6	7.42	0.0406
E9BTF9	Putative 40S ribosomal protein S18	19	28	5	15.04	0.0027
A0A3Q8IWD8	40S ribosomal protein SA	27.5	26	6	5.70	0.0067
E9BK95	Putative ribosomal protein S26	12.8	22	2	15.26	0.0113
E9BJI2	Putative Ribosomal protein S20	13	45	4	17.24	0.0112
E9BQ25	Putative 40S ribosomal protein S3	37.7	10	3	4.80	0.0010
A0A3Q8IDZ4	Putative 60S ribosomal protein L35	15.2	17	2	7.61	0.0017
A0A3Q8I930	Putative 60S ribosomal protein L28	16.3	14	2	108.73	0.0053
E9BBI6	40S ribosomal protein S4	30.6	29	8	5.05	0.0016
E9BFK4	60S ribosomal protein L37a	10.3	28	2	20.92	0.0104
E9BJV7	Putative ribosomal protein S29	6.7	16	1	5.77	0.0191
E9BJZ5	Putative 40S ribosomal protein S17	16.5	21	3	19.50	0.0183
E9BKR3	Putative ribosomal protein L1a	41.1	7	3	17.70	0.0212
E9B840	Putative 60S ribosomal protein L10	24.5	25	5	8.38	0.0346
E9BKE3	40S ribosomal protein S14	15.6	8	1	6.18	0.0222
A0A3Q8IFE1	40S ribosomal protein S19-like protein	18.1	22	1	10.02	0.0065
A0A3Q8ILT7	60S ribosomal protein L2, putative	28.3	15	5	11.38	0.0058

Table 3.

List of interacting proteins belonging to the category ‘Protein folding/ sorting/ degradation’ (map04141) and ‘proteasome’ (map03050)

Accession	Description	MW (kDa)	Coverage (%)	# Unique peptides	Fold change (TCP1γ /PI)	P value
Folding, sorting, and degradation
E9BID4	Heat shock protein 70-related protein (HSP70.4)	70.4	25	12	12.28	0.0042
A0A3Q8IEN5	Heat shock protein 83	80.4	26	17	46.58	0.0134
E9BF40	Putative DnaJ protein (DnaJ3)	49.7	15	5	5.51	0.0126
E9BEY9	Putative ring-box protein 1	12.1	12	1	9.15	0.0028
E9BL53	Putative heat shock protein 20	17.5	17	2	18.88	0.0147
E9BKA7	Putative protein transport protein Sec13	37.8	7	2	5.92	0.0017
E9BJK2	Putative glucose-regulated protein 78	71.8	9	5	45.11	0.0059
E9BI76	Putative protein disulfide isomerase	40.8	11	4	6.39	0.0018
Proteasome
E9BBH3	Putative proteasome regulatory ATPase subunit 2	49.3	4	2	15.24	0.0039
E9BFR3	Putative proteasome regulatory ATPase subunit 1	49	14	5	6.57	0.0018
A0A3S7WX14	Putative proteasome regulatory ATPase subunit 5	49.5	2	1	5.87	0.0041
E9BJ69	Putative proteasome regulatory non-ATPase subunit 3	37.8	4	1	10.01	0.0075
E9BJQ6	Putative proteasome regulatory non-ATPase subunit 2	107.7	3	2	5.66	0.0054
E9B7I1	Putative proteasome regulatory non-ATPase subunit 6	59	8	3	17.60	0.0027
E9BP47	Putative proteasome regulatory non-ATPase subunit	46.4	9	3	21.56	0.0022
A0A3S7WWF6	Proteasome regulatory non-ATPase subunit 5	54.2	6	3	7.24	0.0173

Table 4.

List of top 20 interacting partners of LdTCP1γ classified as uncharacterized proteins

Accession	Description	MW (kDa)	Coverage (%)	# Unique peptides	Fold change (TCP1γ /PI)	P value
A0A3S7X2E5	Uncharacterized protein	67.8	5	3	44.18	0.000
E9BEP7	Uncharacterized protein	81.8	7	4	43.35	0.035
A0A3Q8IG34	Uncharacterized protein	39.2	5	2	36.85	0.048
E9BM30	Uncharacterized protein	13.1	16	1	34.18	0.000
E9BST2	Uncharacterized protein	31.9	9	2	25.71	0.014
E9BHY4	Uncharacterized protein	34.4	7	2	25.57	0.048
A0A3Q8IJM8	Uncharacterized protein	66.6	2	1	25.20	0.001
E9BS23	Uncharacterized protein	35.8	7	2	21.98	0.001
E9BJ94	Uncharacterized protein	37.8	14	4	20.77	0.012
E9BQ11	Uncharacterized protein	45	19	5	19.78	0.000
E9BBW7	Uncharacterized protein	21	17	3	19.47	0.004
E9BKD7	Uncharacterized protein	35.1	13	3	19.29	0.013
E9BQ63	Uncharacterized protein	35.7	9	2	18.78	0.005
A0A3S5H570	Uncharacterized protein	12.4	31	3	18.54	0.002
E9BHF9	Uncharacterized protein	32.4	18	4	17.24	0.010
E9B9E8	Uncharacterized protein	211.9	4	5	16.23	0.019
A0A3Q8ID50	Uncharacterized protein	77.5	2	1	15.50	0.002
A0A3Q8IE86	Uncharacterized protein	60	3	1	15.44	0.005
E9BLX5	Uncharacterized protein	52.5	8	3	14.72	0.031
E9B9G0	Uncharacterized protein	39.5	7	2	14.31	0.042

Validation of proteomics data by immunoblot analysis

Figure 4 depicts western blot analysis of co-IP pullouts to confirm the interaction of HSP90, HSP70, Trypanothione reductase (TR), Tryparedoxin peroxidase (TxnPx), alpha-tubulin, TCP1δ, and TCP1ε with LdTCP1γ protein (Fig. 4). Interestingly, all these proteins were significantly enriched in the LdTCP1γ-co-IP fraction (Fig. 4). In contrast, none of these proteins were detected in the pre-immune control fraction (Fig. 4). The total cell lysate was taken as a positive control (Fig. 4).

Fig. 4.

Validation of the proteomics data by western blot of co-IP assays employing LdTCP1γ antibody and rabbit pre-immune sera (control). anti-HSP90, anti-HSP70, anti-TR, anti-TCP1γ, anti-TCP1δ, anti-TCP1ε, anti-TxnPx, and anti-α-tubulin antibodies are used for detection of respective proteins in the co-IP samples

LdTCP1γ refolds partially denatured TR

Proteomics and western blot analysis identified L. donovani TR protein as one of the interacting partners of LdTCP1γ. Thus, to confirm TR as a substrate of LdTCP1γ homo-oligomeric complex, we tested whether the protein complex is able to refold denatured LdTR. Initially, LdTR was partially denatured with guanidinium hydrochloride (GdmCl) and diluted with refolding buffer with recombinant LdTCP1γ as described in the Methods section. It has previously been demonstrated that complete denaturation of LdTR by GdmCl led to the formation of aggregates that could not be refolded back to active/native LdTR (Rai et al. 2009); hence, partially denatured LdTR was employed as substrate for LdTCP1γ refolding activity. Denatured LdTR exhibited ~ 2-folds (P < 0.001) less activity as compared with native TR. In the presence of 0.5 µM and 1 µM LdTCP1γ, the activity of denatured TR was restored by 25.38% (P < 0.05) and 90.37% (P < 0.001), respectively (Fig. 5).

Fig. 5.

Refolding assay of denatured TR in presence of LdTCP1γ. The data are representative of the result of three independent experiments. The error bars represent SD. ***P < 0.001; **P < 0.01; *P < 0.05; ns, not significant

LdTCP1γ abundance affects the TR activity in L. donovani promastigotes

Figure 6 depicts changes in the cellular activity of L. donovani TR enzyme due to modulated expression of LdTCP1γ. Interestingly, LdTCP1γ single-allele replacement mutant (Dd8^+/−) cells with reduced expression of LdTCP1γ showed significantly decreased LdTR activity (~ two fold, P < 0.01), and overexpression transfectants (Dd8^++/++) exhibited a two fold increase (P < 0.01) in LdTR activity (Fig. 6). In addition, the add-back mutant parasite exhibited similar LdTR activity as that of wild-type parasites (Fig. 6).

Fig. 6.

Effect of differential expression of LdTCP1γ on TR activity of the parasite. The data are representative of the result of three independent experiments. The error bars represent SD. ***P < 0.001; **P < 0.01; *P < 0.05; ns, not significant

Discussion

T-complex polypeptide-1 (TCP1) is a chaperonin protein, known to fold various cytosolic proteins like actin and tubulin in eukaryotes (Sternlicht et al. 1993). Eukaryotic TCP1 chaperonin forms a double-toroidal ring complex of eight subunits each. In Leishmania, only the gamma subunit was cloned and functionally characterized, which forms a homo-oligomeric complex and interacts with both actin and tubulin (Bhaskar et al. 2015; Bhaskar et al. 2012). In S. cerevisiae, about 10% of cytosolic proteins are known to flux through TCP1 indicating the crucial role of this protein complex in various cellular processes (Yam et al. 2008). Therefore, it could be hypothesized that LdTCP1γ complex may also affect many cellular processes in the Leishmania parasite. A mass spectrometry-based method provides a solid platform for mapping protein–protein interaction networks on a large scale (Ewing et al. 2007). Protein–protein interaction network analysis offers information about the function of proteins and is also useful for identifying signaling pathways (Alberts 1998). In the present study, we explored the interactome of LdTCP1γ using co-immunoprecipitation followed by LC–MS/MS–based label-free quantification proteomics.

The interacting partners of LdTCP1γ can be its substrate proteins, chaperones or co-chaperonins, enzymes that modulate the activity of the TCP1 complex, and the proteins of the cross-talking pathways such as protein synthesis and degradation. Out of the 1240 identified proteins, 719 proteins with P < 0.05 and > 4.5-fold change compared with pre-immune control samples were selected as potential interacting partners (Table S1). Interestingly, the presence of all the eight subunits homologous to eukaryotic TCP1 hetero-oligomeric complex (i.e., α, β, γ, δ, ε, ζ, η, and θ) was observed in the pulled out protein complex. This is the first demonstration of the possible presence of hetero-oligomeric complex in Leishmania parasite (Fig. 3). Several studies have demonstrated that TCP1 may function as a homo-oligomer of a single subunit or hetero-oligomers with the mixture of two or more subunits or even a single free subunit in the eukaryotic cell (Roobol and Carden 1999; Echbarthi et al. 2018; Spillman et al. 2017; Bhaskar et al. 2015). Since the abundances of various subunits are dissimilar from each other as shown in the proteomics analysis (Table S1), further studies are required to confirm the presence of hetero-oligomers of two or more subunits in addition to the homo-oligomers of LdTCP1γ in the Leishmania parasite.

To understand the complex interaction network, enrichment and pathway analysis was performed to link LdTCP1γ PPIs to putative biological functions and pathways. The ‘Metabolic pathway’ has emerged as one of the largest categories of proteins that were highly enriched in LdTCP1γ-co-IP assay samples (Figs. 2c and 3). Proteins identified in this category may require LdTCP1 to fold correctly and achieve their native state after synthesis. Some of the identified proteins (Table 1 and Fig. 3) like acetyl-CoA carboxylase, aldolase (fructose-1–6-bisphosphate), ATP synthase subunit beta, cytochrome-c oxidase assembly protein, enolase, glyceraldehyde-3-phosphate dehydrogenase, lactate dehydrogenase, and peroxiredoxin have been shown previously to interact with human and yeast TCP1 chaperonin protein (Wang et al. 2020). Acetyl-CoA carboxylase an important enzyme of the fatty acid synthesis pathway is essential for infectivity of bloodstream Trypanosoma brucei parasite in the mouse model (Vigueira and Paul 2011). Moonlighting functions have been reported for Leishmania aldolase and enolase enzymes apart from their role in the glycolysis/gluconeogenesis pathway. Leishmania aldolase expression in RAW267.4 macrophages was shown to attenuate iNOS induction by IFN-γ (Nandan et al. 2007). Leishmania enolase can bind to plasminogen or plasminogen receptors and facilitates parasite entry into host macrophages (Maldonado et al. 2006; Vanegas et al. 2007). Both aldolase and enolase have been reported in exosomes that are secreted by the parasite to establish early infection in host macrophages (Silverman et al. 2010a; Silverman and Reiner 2011). Glyceraldehyde-3-phosphate dehydrogenase, another enzyme of the glucose metabolic pathway, was shown as a promising drug target in Leishmania (Hannaert et al. 1992). Inhibition of ATP synthases and cytochrome c oxidase complex in Leishmania demonstrated bioenergetic dysfunction of mitochondria being lethal for parasites (Mondal et al. 2014; Luque-Ortega and Rivas 2007). Leishmania cytosolic peroxiredoxin has been shown to protect from reactive oxygen species (ROS) produced by the mammalian immune cells (Iyer et al. 2008). Cytoplasmic peroxiredoxin was found to be associated with the metastatic phenotype of L. Viannia guyanensis, suggesting their role in protection against host microbicidal responses affecting their survival and virulence inside the host cell (Acestor et al. 2006).

LdTCP1γ was found to interact with proteins involved in the biosynthesis of arginines like arginase, glutamamyl carboxypeptidase, and NAD-specific glutamate dehydrogenase (Table 1). Leishmania arginase is the first enzyme of the polyamine pathway, which contains the precursors arginine and ornithine as well as the polyamines: putrescine and spermidine (Ilari et al. 2015). The hypusination and activation of eukaryotic initiation factor 5A (eIF5A) and the production of trypanothione, which is required for oxidative stress protection, are crucial downstream reactions (Ilari et al. 2015). Recently the relevance of polyamines and trypanothione in parasite growth and infectivity has been demonstrated, establishing the pathway as a promising therapeutic target (Roberts and Ullman 2017). Leishmania lacks de novo purine synthesis mechanism as that of mammals and thus it depends on the host to survive (Moreira and Murta 2016). Nucleoside diphosphate kinase is an enzyme of the purine synthesis pathway which catalyzes the transfer of phosphate from nucleoside triphosphate to nucleoside diphosphate (Kolli et al. 2008). In addition to the normal housekeeping functions, Leishmania nucleoside diphosphate kinase has been also found to prevent ATP-mediated lysis of macrophages and thus maintain host cell integrity to the benefit of the parasite (Kolli et al. 2008). Proteomic analysis of co-IP pull-out proteins indicated the interaction of enzymes of sterol pathway with LdTCP1γ (Table 1). Cell membrane sterols maintain membrane fluidity and contribute to membrane organization (de Souza and Rodrigues 2009). Similar to fungi, cell membranes of the Leishmania parasite contain ergosterol and ergosterol-like sterols instead of cholesterol in mammalian cells (de Souza and Rodrigues 2009). Sterol C-24 reductase is an important enzyme of the ergosterol biosynthetic pathway of L. donovani which converts ergostatetraenol to ergosterol. Sterol C-24 reductase has been also shown as a drug target to treat visceral leishmaniasis (Tabrez et al. 2021). Thus, LdTCP1γ interacting partners were not only involved in the essential biological process of the parasite but also play a crucial role in virulence and pathogenicity. This is in accordance with our previous report which showed that LdTCP1γ null-mutants were unable to survive (Yadav et al. 2020b).

The next enriched pathway category is ‘Ribosome’, and all the identified proteins were the structural constituents of the ribosome (Table 2, Figs. 2c and 3). Interestingly, the biological processes GO term annotation analysis revealed that about 39% of annotated proteins were from translation (Fig. 2a). Furthermore, 51% of annotated proteins are structural constituents of the ribosome as shown by molecular function GO term enrichment analysis (Fig. 2b). This is not surprising because the TCP1 was previously shown to be closely associated with the translational apparatus and helps the translating polypeptide to fold as soon as it emerges from the ribosome in yeast (McCallum et al. 2000).

Proteasome was another enriched pathway shown in string enrichment analysis (Fig. 2c). Cooperation between the molecular chaperones and proteasomal degradation machinery occurs during the protein synthesis and refolding of denatured proteins because some proteins cannot attain their native state spontaneously (Esser et al. 2004). Thus, folding and degradation pathways form the protein quality control system of the eukaryotic cell (Esser et al. 2004). This system selectively recognizes misfolded proteins, associates them with molecular chaperones, and targets them for proteasomal degradation (Park et al. 2007). Chaperones like HSP70 have been shown to form a coordinated network with co-chaperones and the proteasome pathways in yeast (Kandasamy and Andreasson 2018). TCP1 complex interactome analysis in yeast showed many of the 20S proteasome subunits (Yam et al. 2008). Further, a computational analysis of the interactome of yeast and human TCP1 based on physical and experimental interactions showed enrichment of proteins from the proteasome pathway (Narayanan et al. 2016).

The next enriched pathway was protein folding and trafficking which mainly included various molecular chaperones, particularly HSP70.4, HSP83, HSP40 (DnaJ3), HSP20, and GRP78 (Table 3) (Requena et al., 2015). HSP70 is considered a central player in the protein quality control system. It cooperates with other molecular chaperone like chaperonins, HSP90, small heat shock proteins, and HSP100 and with ubiquitin/proteasomal degradation system, forming a versatile functional network in eukaryotes (Rosenzweig et al. 2019). Hsc70 (HSP70 family protein) interaction with the apical region of TCP1β subunits was reported previously suggesting delivery of the unfolded substrate by Hsc70 to the substrate-binding region of TCP1 (Cuellar et al. 2008). The cooperation of TCP1 and HSP70 was demonstrated, suggesting partitioning of substrate protein among them (Boudiaf-Benmammar et al. 2013). This further suggests that the TCP1 chaperonin may work in coordination with other chaperones. Several LdTCP1γ interacting proteins were classified as uncharacterized protein because they lacked identifiable orthologues in other organisms (Table 4 and Table S1). However, these uncharacterized proteins exhibited highly significant fold change (14–43 folds) (Table 4).

LdTCP1γ has been shown to protect the parasite from miltefosine-induced apoptosis (Yadav et al. 2020a). Further, differential regulation of LdTCP1γ has been shown to influence the cellular level of thiols (Yadav et al. 2020a) suggesting a role of LdTCP1γ in the regulation of thiol redox machinery of the parasite. In accordance, LdTCP1γ was found to interact with enzymes of the trypanothione/glutathione pathway like trypanothione reductase (TR), tryparedoxin peroxidase (TxnPx), mitochondrial peroxiredoxin, and glutathione peroxidase (Table 1). This interaction was further confirmed by evaluating refolding of trypanothione reductase, one of the interacting redox pathway proteins. Interestingly, LdTCP1γ was able to refold denatured TR to its active state (Fig. 5). Further, modulation of LdTCP1γ expression in parasites also modulates TR activity (Fig. 6). The data suggest that TR may require cytosolic LdTCP1γ chaperonin for maintaining active conformation. Thus, the present study also provides mechanistic insight into the association of LdTCP1γ with the redox pathway and hence miltefosine resistance.

Conclusion

Thus, taken together, LdTCP1γ interactome showed a diverse set of proteins affecting many cellular activities. LdTCP1γ interaction with ribosomal proteins, proteasome regulatory subunits, enzymes involved in cellular metabolism, and other chaperones suggest an interlinked network of protein operating in the cell. This is the first-ever report to reveal the functional aspects of the interacting partners of TCP1 chaperonin protein in any of the protozoa parasites till now. Thus, knowledge of the interactome of LdTCP1γ will provide a novel insight into the functional role of LdTCP1γ in L. donovani parasite.

Supplementary Information

Below is the link to the electronic supplementary material.

Abbreviations

CCT

Chaperonin-containing TCP1

HSP

Heat shock protein

LdTCP1γ

Leishmania donovani T-complex protein-1 gamma subunit

Author contributions

NG conceived the study, designed the experiments, analyzed the data, wrote the manuscript, and performed overall supervision. SY performed the experiments, analyzed data, and wrote the manuscript. JSM contributed to LC–MS/MS analysis. KR contributed to animal experiments. AA and DCB performed data analysis and writing of the manuscript.

Data availability

Data will be made available upon reasonable request.

Declarations

Ethics approval

Animal studies were carried out in accordance with the guidelines approved by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Ministry of Environment and Forest, Government of India. The protocol was authorized by the Institutional Animal Ethics Committee (IAEC) of CSIR-Central Drug Research Institute (IAEC/2018/F-37).

Consent to participate

The authors agree to this.

Consent for publication

The authors agree to this.

Competing interests

The authors declare no competing interests.