Abstract

Per-Arnt-Sim (PAS) domains are structurally conserved and present in numerous proteins throughout all branches of the phylogenetic tree. Although PAS domain-containing proteins are major players for the adaptation to environmental stimuli in both prokaryotic and eukaryotic organisms, these types of proteins are still uncharacterized in the trypanosomatid parasites, Trypanosome and Leishmania. In addition, PAS-containing phosphoglycerate kinase (PGK) protein is uncharacterized in the literature. Here, we report a PAS domain-containing PGK (LmPAS-PGK) in the unicellular pathogen Leishmania. The modeled structure of N-terminal of this protein exhibits four antiparallel β sheets centrally flanked by α helices, which is similar to the characteristic signature of PAS domain. Activity measurements suggest that acidic pH can directly stimulate PGK activity. Localization studies demonstrate that the protein is highly enriched in the glycosome and its presence can also be seen in the lysosome. Gene knockout, overexpression and complement studies suggest that LmPAS-PGK plays a fundamental role in cell survival through autophagy. Furthermore, the knockout cells display a marked decrease in virulence when host macrophage and BALB/c mice were infected with them. Our work begins to clarify how acidic pH-dependent ATP generation by PGK is likely to function in cellular adaptability of Leishmania.

Introduction

Human pathogen Leishmania are digenetic parasites, whose life cycle involves two hosts: the vertebrate macrophage and the sand fly vector. The transformation of the parasite from its promastigote stage (inside the midgut of the sand fly) to the amastigote stage (inside the vertebrate macrophage) requires several significant cellular remodeling, which in turn are heavily dependent on the environmental cues [1]. One of the key cellular remodeling processes during this transformation is the modulation of the glycosome [2], wherein the activity and the type of different glycosomal enzymes change considerably. Even, the number of glycosomes per cell changes and this is regulated by a co-ordinated process called pexophagy (autophagy of glycosomes) [3]. Usually, the number of glycosomes in a promastigote is more than that of in an amastigote and this turn-over of the glycosome has been implicated to play a very important role in cellular differentiation [2].

Per-Arnt-Sim (PAS) domains are widely present in proteins of all kingdoms of life. The PAS domains are usually made up of 100–120 amino acids and are bound to a wide range of enzymatic and nonenzymatic regulatory modules, which participate in various cellular signaling pathways [4]. PAS domains carry out diverse functions within sensory proteins by transferring signals [5] or facilitating protein/protein interaction [6] as well as by directly sensing environmental stimuli [7]. Depending on the structure, small molecules/ions can bind to the PAS domain can either serve as a direct signal [8] or can bind to a cofactor that allows the perception of signals like dissolved gases [9], redox potential [10,11] and visible light [12]. On the basis of genome studies, recently a comprehensive review on PAS domains in kinetoplastids has been published in the literature [13]. Despite the diverse function of PAS domains in both Prokarya and Eukarya, no PAS domain-containing protein has been characterized from the Kinetoplastida group.

Phosphoglycerate kinase (PGK) catalyzes the reversible conversion of ADP and 1,3-bisphosphoglycerate to ATP and 3-phosphoglycerate. Besides, it is considered as a moonlighting protein. A group of biologists have already reported that Leishmania contains two PGKs, one localized in the cytosol (PGKB) and the other localized in the glycosome (PGKC) and they are expressed in both the amastigote and the promastigote forms [14]. The cytosolic and glycosomal PGK are expressed as 80% and 20% of the total PGK activity, respectively [14,15]. PGKC has a 62 residue C-terminal extension with a peroxisomal targeting signal (PTS), which is responsible for importing this isoenzyme to the glycosomes [16].

In this manuscript, we have characterized a novel PAS domain-containing PGK protein from Leishmania major (LmPAS-PGK), which contains an N-terminal regulatory PAS domain that is linked to a C-terminal catalytic PGK domain. To understand the biochemical function of this protein, we cloned, expressed, and characterized the LmPAS-PGK protein. Surprisingly, our data revealed that the PAS domain of LmPAS-PGK primarily regulates the PGK activity with respect to pH changes. Experimental results provide evidence for the existence of the LmPAS-PGK, which can catalyze optimal ATP synthesis at an acidic pH 5.5 and it is localized in both the glycosome and the lysosome. In addition, we present, for the first time, LmPAS-PGK null mutants have more autophagosomes and they have less infectious property with respect to macrophage infection as well as cutaneous lesions in BALB/c mice.

Experimental procedures

Parasite culture

Leishmania major wild-type parasites (strain 5ASKH) were cultured at 22°C in M199 medium (Invitrogen) supplemented with 40 mM HEPES, 200 µM adenine, 1% penicillin–streptomycin (Invitrogen) (v/v), 50 µg/ml gentamicin (Abbott), and 10% heat-inactivated fetal bovine serum (FBS, Invitrogen).

Animal ethics statement

All BALB/c mice were obtained from and maintained in our institutional animal facility (Kolkata, India). The studies were approved by CSIR-IICB Animal Ethical Committee (Registration no. 147/1999, CPCSEA), registered with Committee for the purpose of Control and Supervision on Experiments on Animals (CPCSEA), Govt. of India, and BALB/c mice were handled according to their guidelines.

Cloning of L. major PAS-PGK

Genomic DNA from L. major promastigotes was isolated by a genomic DNA isolation kit (Qiagen). The PCR amplification was carried out by using primers 1 and 2 (Supplementary Table S1) for cloning of full-length proteins and primers 3 and 4 (Supplementary Table S1) for cloning of Δ115 PAS-PGK. The PCR products from two separate reactions were purified and cloned into the BamHI and HindIII sites of pTrcHisA (Invitrogen) for full-length LmPAS-PGK and the BamHI and HindIII sites of pET28a (Novagen) for Δ115 LmPAS-PGK.

Overexpression of LmPAS-PGK with or without N-terminal RFP tag

LmPAS-PGK ORF was amplified using primers 5 and 6 (Supplementary Table S1) and cloned into the SmaI- and BamHI digested pXG-B2863 vector. The primers 7 and 8 (Supplementary Table S1) were used for making PCR product in PAS-PGK-RFP-fused overexpression construct and cloned into the BglII/NotI site of pNUS-mRFPnD vector. Transformation of the LmPAS-PGK-containing vectors in Leishmania cells was performed by electroporation as described earlier [17]. Overexpressed cells with or without RFP were maintained at 100 µg/ml blasticidin (Invitrogen) or 200 µg/ml neomycin (Roche), respectively.

Generation of stable knockout strain for LmPAS-PGK alleles

Modified pXG-Neo and pXG-Hyg vectors were used to generate the knockout constructs of LmPAS-PGK gene. Primers 9 and 10 (Supplementary Table S1) were used for amplifying 1.0 kbp 5′-flank, and primers 11 and 12 (Supplementary Table S1) were used for amplifying 1.0 kbp 3′-flank of the gene. Both 5′-flank and 3′-flank DNA fragments were cloned on either side (HindIII/SalI and SmaI/BamHI) of neomycin and hygromycin gene of pXG-Neo and pXG-Hyg vectors, respectively [18]. Both constructs were then digested with HindIII and BamHI to get linear fragments of the gene deletion constructs LmPAS-PGK::NEO and LmPAS-PGK::HYG, which were transfected into L. major sequentially. Knockout cells were maintained in 50 µg/ml neomycin (Roche) and 100 µg/ml hygromycin drug (Roche).

Gene knockouts were confirmed by PCR using primers 13 and 14 (Supplementary Table S1) as internal to the gene. Primers 15 and 16 (Supplementary Table S1) were used as external to the gene. KO cells were further confirmed by Western blotting.

Complementation of LmPAS-PGK in null mutants

To restore LmPAS-PGK in the knockout parasites, LmPAS-PGK ORF was amplified by PCR using the primers 17 and 18 (Supplementary Table S1). The amplified product was cloned at the same site of pXG-PHLEO vector and the plasmid DNA was transfected into the knockout promastigotes. Transfected promastigotes were finally maintained in the presence of 60 µg/ml neomycin, 100 µg/ml hygromycin and 10 µg/ml phleomycin drug. Complimentary clones were confirmed by measuring LmPAS-PGK expression by Western blot analysis with rabbit anti-LmPAS-PGK antibody (1:50).

N-terminal RFP-fused cysteine peptidase B (RFP/CPB) overexpression system was constructed for use in Leishmania

Primers 19 and 20 (Supplementary Table S1) were used to amplify the ORF of L. major CPB (LmjF.08.1010) by PCR. The amplified portion was cloned into the BglII/NotI sites of pNUS-mRFPnD vector. The DNA was then transfected in L. major by electroporation. Overexpressed cells were maintained at 100 µg/ml blasticidin (Invitrogen).

N-terminal FLAG-fused vacuolar proton pyrophosphatase 1 (FLAG/VP1) overexpression system was constructed for use in Leishmania

Primers 21 and 22 (Supplementary Table S1) were used to amplify the ORF of L. major vacuolar proton pyrophosphatase 1 (LmjF.31.1220) by PCR. The amplicon was cloned into the SmaI/BamHI sites of pXG-B2863 vector. The DNA was then electroporated into the L. major cells. Overexpressing cells were maintained at 100 µg/ml neomycin (Roche).

RFP/CPB and FLAG/VP1-fused overexpression system were constructed for use in Leishmania

The FLAG tag VP1-containing pXG-B2863 vector was transfected into the RFP/CPB-fused overexpression system in Leishmania promastigotes. Transfected promastigotes were finally maintained in the presence of 100 µg/ml neomycin and 100 µg/ml blasticidin drug.

RFP/ATG8-fused overexpression system was constructed for use in Leishmania

Primers 23 and 24 (Supplementary Table S1) were used to amplify the ORF of L. major ATG8 gene (LmjF.19.1630) by PCR. The amplified portion was cloned into the BglII/NotI site of pNUS-mRFPnD vector generating an N-terminal RFP-fused ATG8. The DNA was then transfected in CT, OE, KO and CM cell type separately by the electroporation. Both RFP/ATG8 overexpressing CT and OE cells were maintained at 100 µg/ml neomycin (Roche) and 100 µg/ml blasticidin (Invitrogen). The RFP/ATG8 overexpressing KO cells were cultured in the presence of neomycin (50 µg/ml), hygromycin (100 µg/ml) and blasticidin (100 µg/ml). RFP/ATG8-overexpressing CM cells were maintained in the presence of 60 µg/ml neomycin, 100 µg/ml hygromycin, blasticidin (100 µg/ml) and 10 µg/ml phleomycin drug.

Expression and purification of protein

Recombinant LmPAS-PGK were transformed into Escherichia coli BL21 (DE3) and were grown overnight in 50 ml of Luria–Bertani broth containing 200 µg/ml ampicillin (Sigma) at 37°C. The overnight grown culture was then inoculated in 500 ml of terrific broth. When the culture reached an absorbance of around 0.6–0.8 at 600 nm, 0.5 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) was added and bacteria were further grown at 25°C for ∼18 h. Cells were then harvested by centrifugation at 8000×g for 10 min and washed two times with 1× PBS. The pellet of a 500 ml culture was resuspended in 5 ml of 50 mM Tris buffer (pH 7.5) containing 250 mM NaCl, 10% glycerol, a protein inhibitor mixture tablet without EDTA (Roche) and PMSF (Sigma). The resuspended solution was kept in ice for ∼45–60 min and the cells were freeze-thawed in liquid nitrogen followed by a 20-sec pulse sonicator with 40-sec rest on ice in-between. Then the lysate was centrifuged at 14 000×g for 60 min at 4°C. The cell-free supernatant, also called the crude extract was loaded onto a Ni2+-NTA column. The column was washed with equilibrium buffer containing 50 mM Tris buffer, pH 7.5 containing 250 mM NaCl, 10% glycerol, followed by the same buffer containing 50 mM imidazole and eluted with equilibrium buffer containing 250 mM imidazole and dialyzed three times against 20 mM Tris buffer (pH 7.5) having 10% glycerol.

The purification of Δ115LmPAS-PGK was carried out in the same way as above except recombinant Δ115 LmPAS-PGK after transformation into Escherichia coli BL21 (DE3) were grown in Luria–Bertani broth containing 50 µg/ml kanamycin (SRL) and after induction with IPTG, it was further grown at 16°C for ∼18 h. Molecular weight and purity of both the proteins were confirmed by 13% SDS–PAGE.

Size-exclusion chromatography

LmPAS-PGK was studied by gel exclusion chromatography at 28°C using a column namely Biosuite™ 250 (Waters) in an HPLC system preequilibrated with 50 mM potassium phosphate buffer pH 7.5 containing 150 mM NaCl, with a flow rate 0.8 ml/min at A280. Column calibration was done using standard protein mixture of glutamate dehydrogenase (290 kDa), lactate dehydrogenase (142 kDa), enolase (67 kDa), myokinase (32 kDa) and cytochrome c (12 kDa).

Enzyme assay using UV-visible spectroscopy

All kinetic studies of both LmPAS-PGK and Δ115LmPAS-PGK were performed at 25°C on Shimadzu UV-2550 spectrophotometer using quartz cuvette of 1.0-cm path length. The assay mixture was composed of 40 mM TEA-HCl buffer (pH 7.5) (Amresco), 0.15 mM NADH (Merck), 0.8 mM MgSO4, 1 mM ATP (Sigma), 2.5 mM 3-phosphoglyceric acid (Sigma) and 0.04U of rabbit muscle glyceraldehyde 3-phosphate dehydrogenase (GAPDH, Sigma). After incubating in a spectrophotometer at 25°C for 5 min to achieve temperature equilibrium, the reaction was initiated with the addition of pure enzyme. Absorbance from 0 to 5 min was recorded. NADH oxidation was measured as loss of the absorbance at 340 nm. The concentration of NADH was determined from ɛ340 = 6.2 × 103 M−1 cm−1.

The different PAS domain ligands were added in the assay mixture individually in their effective concentration.

Determination of the pH-dependent enzyme activity

To determine the pH-optimum of LmPAS-PGK and Δ115LmPAS-PGK, kinetic studies of both the proteins were done using the following buffers (50 mM): acetate (pH 3.5–5.5), MES and phosphate buffers (pH 5.5–6.5), Tris–HCl (pH 6.8–8.8), TEA-HCl (pH 7.5) under standard conditions. The pH was adjusted at room temperature. Shimadzu UV-2550 spectrophotometer with quartz cuvette of 1.0-cm path length was used.

Fluorescence quenching

To determine the folding properties of full-length LmPAS-PGK under different pH conditions, fluorescence quenching of tryptophan residue was observed. A scan from 320 nm to 380 nm was taken after exciting the protein at 295 nm using buffers of different pH. The buffers used were acetate buffer (pH 3.5–5.5), phosphate buffer (pH 6.0–7.0), TEA-HCl (pH 7.5), Tris–HCl (pH 8.0–9.0). F-7000 FL Spectrophotometer, Hitachi was used with 1.0 cm path length cuvette.

Production of polyclonal antibodies against LmPAS-PGK

Polyclonal antibodies against the purified recombinant PAS-PGK (20 µg) was raised by subcutaneous injection in 6-month-old female rabbit using Freund's complete adjuvant (Sigma). This was followed by three booster doses of recombinant PAS-PGK (15 µg) with incomplete adjuvant (Sigma) at 2-week intervals. The rabbit was bled 2 weeks after the last booster and sera were collected and used for western blot analysis.

Western blot analysis

Proteins were resolved on 13% SDS–PAGE and then transferred on nitrocellulose membrane (GE Healthcare) by Bio-Rad semidry apparatus. After 1 h of blocking in 5% BSA, the membrane was incubated overnight with antiserum against recombinant LmPAS-PGK protein at a dilution of 1:50 at 4°C. The membrane was washed with 1× TBS containing 0.1% Tween20 (TBS-T) and then incubated in alkaline phosphatase (AP)-conjugated anti-rabbit secondary antibody (1:15 000) of Sigma. NBT/BCIP solution from Roche was used for band detection. α-Tubulin was used as endogenous control and AP-conjugated anti-mouse secondary antibody (1:15 000) was used against α-tubulin.

In case of determining RFP/ATG8 and RFP/ATG8-PE, the proteins were resolved on 10% SDS–PAGE containing 6 M urea and then transferred on PVDF membrane (Merck Millipore) by Bio-Rad semidry apparatus. The membrane was blocked by 5% BSA for 30 min and incubated overnight with anti-rabbit RFP antibody (Thermo Scientific) at a dilution of 1:1000 at 4°C. The membrane was washed with 1× TBS containing 0.1% Tween 20 (TBS-T) and then incubated with AP-conjugated anti-rabbit secondary antibody (1:15 000) of Sigma. NBT/BCIP solution (Roche) was used for band detection. Wortmannin (Sigma) and bafilomycin A1 (Sigma) were used as controls at concentrations of 10 µM and 50 nM, respectively.

Co-localization of the protein expressed from the recombinant gene pNUS/PGK-RFP with the acidic compartments

107 mid-log phase pNUS/PGK-RFP overexpressing L. major cells were washed twice with PBS and then resuspended with 1 ml PBS. 3 µM of lysotracker green (Molecular Probe, Invitrogen) was added and incubated for 30 min at 28°C. Then the cells were washed with PBS and fixed on the poly-l-lysine-coated slides. Finally, they were stained with DAPI and mounted with antifade mounting media, and observed under a confocal microscope (Leica). The wavelength used were DAPI (Exλ = 350 nm, Emλ = 470 nm), RFP (Exλ = 535 nm, Emλ = 615 nm), lysotracker green (Exλ = 504, Emλ = 511 nm).

Co-localization of the protein expressed from the recombinant gene pNUS/PGK-RFP with glycosome

107 mid-log pNUS/PGK-RFP overexpressing L. major cells were washed thrice with 1× PBS and then fixed on poly-l-lysine-coated slides. The slides were dipped in a solution containing 1% Triton X-100 and 50 µg/ml RNase A (Calbiochem). Following the blocking with 1.5% BSA for 2 h, the slides were incubated with anti-rabbit GAPDH antibody (1:800) for overnight at 4°C. The next day, the slides were washed with 1× PBS and incubated with Alexa Fluor 488-conjugated anti-rabbit secondary antibody (Thermo Scientific) at a dilution of 1:2000 for 2 h. Finally, they were stained with DAPI and mounted on a poly-l-lysine-coated slide with antifade mounting media (Invitrogen). The wavelength used for Alexa Flour 488 secondary antibody were Exλ = 488 nm and Emλ = 522 nm.

Co-localization of LmPAS-PGK in the lysosome

107 mid-log pNUS/CBP-RFP overexpressing L. major cells were washed with 1× PBS and then fixed on poly-l-lysine-coated slides. The slides were dipped in a solution containing 1% Triton X-100 and 50 µg/ml RNase A. Following the blocking with 1.5% BSA for 2 h the slides were incubated with anti-rabbit PAS-PGK antibody (1:800) for overnight at 4°C. The next day, the slides were washed with 1× PBS and incubated with Alexa Flour 488 goat anti-rabbit secondary antibody at a dilution of 1:2000 for 1 h. Finally, they were mounted with antifade mounting media (with DAPI).

Co-localization of LmPAS-PGK in the acidocalcisome

The co-localization of LmPAS-PGK with the acidocalcisomal marker VP1 was determined by double immunofluorescence. 107 mid-log pXG/FLAG-VP1 overexpressing L. major cells were washed with 1× PBS and then fixed on poly-l-lysine-coated slides. The slides were dipped in a solution containing 1% Triton X-100 and 50 µg/ml RNase A. Following the blocking with 1.5% BSA for 2 h, the slides were incubated with a mixture of anti-rabbit PAS-PGK antibody (1:800) and anti-mice FLAG antibody (Sigma) for overnight at 4°C. The next day, the slides were washed with 1× PBS and incubated with both anti-mice Alexa Fluor 546 (Thermo Scientific) and anti-rabbit Alexa Fluor 488-conjugated secondary antibody at a dilution of 1:2000 for 2 h. Finally, they were mounted with antifade with DAPI. The wavelength used for Alexa Fluor 546-conjugated secondary antibody were Exλ = 546 nm and Emλ = 575 nm.

Co-localization of LmPAS-PGK with mitochondria

107 mid-log phase pNUS/PGK-mRFP overexpressing L. major cells were washed twice with PBS and then resuspended with 1.0 ml PBS. 500 nM of MitoTracker green (Molecular Probe, Invitrogen) was added and incubated for 30 min at 28°C. Then the cells were washed with PBS and fixed on the poly-l-lysine-coated slides. Finally, they were mounted with antifade with DAPI. The wavelength used for MitoTracker green were Exλ = 490 and Exλ = 516 nm.

Isolation of mitochondrial and cytosolic fractions

Isolation of mitochondrial and cytosolic fractions was performed from L. major promastigotes by hypotonic lysis followed by Percoll gradient centrifugation at 4°C [17]. The purity of each fraction was checked by Western blot analysis with organelle-specific marker rabbit anti-(L. major) ascorbate peroxidase (APX, 1:50) antibodies for the mitochondria and rabbit anti-(L. donovani)-adenosine kinase (ADK, 1:50) for the cytosol. The AP-conjugated anti-rabbit secondary antibody (Sigma) was used at a dilution 1:15 000.

Isolation of lysosomal fractions

Lysosome was isolated following the method described by Jinn et al. [19] with slight modifications. Briefly, L. major cells were centrifuged 500×g for 5 min to obtain cell pellets. After the removal of media, pellets were washed with PBS and centrifuged again. The obtained pellets were resuspended in sucrose homogenization buffer (0.25 M sucrose, 20 mM HEPES) containing protease inhibitor (Roche), and lysed by Dounce homogenizer and cell lysis was confirmed under a light microscope. Homogenates were centrifuged again at 500×g for 5 min to eliminate the cell debris. The supernatant was centrifuged at 7000×g for 10 min to eliminate the mitochondrial fraction. The resulting supernatant was centrifuged again at 20 000×g for 60 min to yield the crude lysosomal fraction (CLF) as a pellet. The CLF was washed again with 10 mM CaCl2 and centrifuged at 7000×g for 15 min. The supernatant contains purified lysosome. The purity of lysosomal fraction was checked by Western blot analysis with organelle-specific marker RFP tag CPB. The primary antibody used was anti-rabbit RFP antibody.

Isolation of acidocalcisomal fractions

Acidocalcisome was isolated using the method described by Salto et al. [20] with certain modifications. L. major cells were centrifuged at 500×g for 10 min to obtain cell pellets. The pellets were then washed with PBS and centrifuged. The resultant pellet was resuspended in the lysis buffer comprising of 125 mM sucrose, 50 mM KCl, 4 mM MgCl2, 0.5 mM EDTA, 20 mM K-HEPES, 5 mM dithiothreitol and protease inhibitor cocktail. The cells were completely homogenized by syringe (needle gauge 0.30 × 12.5 mm). The lysate at first was centrifuged at 150×g for 5 min and then at 325×g for 10 min. The supernatant fractions were taken together and centrifuged for 30 min at 11 000×g. The pellet was resuspended in lysis buffer and applied to the 34% step of the discontinuous (20–40% (v/v)) gradient of iodixanol, with gradients of 20, 25, 30, 34, 37, and 40% iodixanol, diluted in lysis buffer. The gradient was centrifuged at 50 000×g for 60 min. The acidocalcisome fraction pelleted on the bottom of the tube and was resuspended in lysis buffer. The purity of acidocalcisomal fraction was checked by Western blot analysis with organelle-specific marker FLAG-tagged vacuolar proton pyrophosphatase 1. The primary antibody used was anti-mice FLAG antibody.

Isolation of glycosomal fractions

Glycosome was isolated following the method described by Colasante et al. [21] with minor modifications. L. major promastigotes were harvested by centrifugation for 10 min at 2000×g, and were washed once in 10 ml of wash buffer (25 mM Tris, 1 mM EDTA, 1 mM DTT, 250 mM sucrose, pH 7.8). After centrifugation, the cell pellet was resuspended in 2 ml homogenization medium (250 mM sucrose, 1 mM EDTA, 0.1% (v/v) ethanol, 5 mM MOPS, pH 7.2) containing protease inhibitor (complete EDTA-free, Roche Applied Science) and was lysed using syringe (needle gauge: −0.30 × 12.5 mm). Cell lysis was confirmed by light microscopy. The cell lysate was centrifuged sequentially for 5 min each at 100×g and 3000×g to remove cell debris and cell nuclei. The resulting supernatant was centrifuged at 17 000×g for 15 min to obtain a glycosomal enriched pellet. This pellet was resuspended in 1.0 ml homogenizing buffer and was loaded on top of a 24 ml linear 20–40% (v/v) optiprep gradient (Sigma), mounted on 50% 2.5 ml optiprep cushion and centrifuged at 170 000×g for 1 h. Nine fractions (1.0 ml each) were collected from the bottom of the tube. They were centrifuged at 30 000×g to obtain the pellet. The pellet was resuspended in homogenizing buffer and again centrifuged at 30 000×g to obtain the purified glycosomal pellet. The purity of the fraction was checked by western blotting using the glycosome-specific GAPDH. The primary antibody used was rabbit anti-(L. major) GAPDH antibody (1:100).

Measurement of pyrophosphatase activity in purified acidocalcisome

Around 58 mg/ml protein equivalent of purified acidocalcisome with and without 0.5% Triton X was incubated in a solution containing 50 mM Tris–HCl (pH 7.5), 10 mM sodium pyrophosphate and 10 mM magnesium chloride for 10 min [22,23]. After 10 min, the detection reagent containing 10% ammonium molybdate in 10 N H2SO4 and 5% ferrous sulfate was added. The resulting solution was incubated again for 10 min at 25°C. Then 500 µl aliquots from each sample is transferred to spectrophotometer cuvette and the absorbance was recorded at 660 nm. Inorganic phosphates were used as standards. A standard curve was prepared by plotting the ΔA660 nm versus concentration of phosphate.

Measurement of peroxidase activity in purified mitochondria

3 mg/ml protein equivalent of purified mitochondria with and without 0.5% Triton X was incubated in a solution containing 50 µM ferrocytochrome c, 50 mM phosphate buffer (pH 7.5), at a final volume of 1.0 ml. The reaction was started by the addition of 0.3 mM H2O2. Ferrocytochrome c oxidation was measured as loss of absorbance at 550 nm. The concentration of oxidized cytochrome c was determined from ɛ550 = 2.1 × 104 M−1 cm−1.

Measurement of ATP

107 cells were lysed by 0.5% Triton X in the presence of 50 µM of ARL 67156 trisodium salt hydrate (Sigma) at 95°C for 1 min. ATP content in the lysate was measured by a luciferin–luciferase bioluminescence assay using the ATP determination kit (Molecular Probes) according to the manufacturer's protocol. Briefly, the samples were added to the reaction mixture containing reaction buffer, D-luciferin, DTT and the enzyme firefly luciferase and the luminescence was measured by a luminometer (Glomax, Promega). ATP concentrations were calculated from the ATP standard curve.

Measurement of glycolytic flux

The glycolytic flux of the CT, OE, CM and KO cell lines was measured by using the Glycolysis Cell-Based Assay Kit (Cayman Chemicals) following the manufacturer's protocol. Briefly, CT, OE, CM and KO cells were grown in M199 media with and without 50 mM inhibitor of glycolysis (2-deoxy d-glucose). 107 cells from each cell type were centrifuged at 3500 rpm for 5 min and the supernatant was collected. 10 µl of supernatant was added to 190 µl of reaction solution containing assay buffer, glycolysis assay enzyme mixture, glycolysis assay cofactor and glycolysis assay substrate. The resulting mixture was carefully loaded onto 96-well plate using a multichannel pipette. After 30 min of incubation, the absorbance was measured at 490 nm using a plate reader. The lactate provided by the manufacturer was used as standard.

Determination of autophagic flux by flow cytometry

107 log phase CT, OE, CM and KO cells from control and starved conditions were washed twice and resuspended in 1× assay buffer containing 2.0 µl of the provided green detection reagent (autophagy detection kit dye, Abcam) and incubated for 30 min at 37°C. The samples were then sorted by flow cytometry (BD FACS LSRFortessa) using the FITC filter (Exλ = 488 nm, Emλ = 517 nm). 10 000 events were measured for every sample. The nutrient starvation was achieved by incubating the cells in 1× PBS at 28°C for 2 h.

Determination of autophagic flux by fluorescence microscopy

107 log phase CT, OE, CM and KO control and nutrient-starved promastigotes were washed twice and resuspended in 1× assay buffer containing 2 µl of the provided green detection reagent (autophagy detection kit dye) and incubated for 30 min at 37°C. The cells were then fixed on poly-l-lysine-coated slides. Finally, they were mounted with antifade mounting media (with DAPI) and observed under a fluorescence microscope (Olympus). The wavelength used were-DAPI (Exλ = 350 nm, Emλ = 470 nm) and autophagy detection kit dye (Exλ = 488 nm, Emλ = 517 nm). The nutrient starvation was achieved by incubating the cells in 1× PBS at 28°C for 2 h.

Determination of the formation of ATG8 puncta

107 mid-log phase CT, OE, CM and KO cells overexpressing RFP/ATG8 from the normal and stressed conditions were washed twice with 1× PBS and fixed on poly-l-lysine-coated slides. They were mounted with antifade mounting media with DAPI and observed under a fluorescence microscope. The wavelength used were-DAPI (Exλ = 350 nm, Emλ = 470 nm) and RFP/ATG8 (Exλ = 535 nm and Emλ = 615 nm). The nutrient starvation was achieved by incubating the cells in 1× PBS at 28°C for 2 h.

Infection in mice

Disease progression was monitored by daily caliper measurement of footpad swelling. Parasite loads in footpad tissue of mice containing CT, OE, CM and KO parasites were determined by limiting dilution assay with slight modifications. Briefly, 1 mg of footpad tissue were sequentially immersed in 70% ethanol, and sterile H2O before homogenization of weighed tissue in M199 media supplemented with gentamicin and penicillin–streptomycin containing 10% heat-inactivated FBS. Each tissue homogenate was serially diluted in the same medium in a 96-well flat-bottom tissue culture plate. The number of viable parasites per milligram of tissue was determined from the highest dilution at which promastigotes could be grown out after up to 10–15 days’ incubation at 22°C.

In vitro promastigote growth profile analysis

106 mid-log phase cells were inoculated in 10 ml of M199 media supplemented with 10% FBS. Growth rates were measured at a 24-h interval by counting cell number in an improved Neubauer chamber (hemocytometer) for 8 consecutive days. Experiments were done in triplicate for each cell type.

Determination of cell viability by flow cytometry

Cell viability was determined by propidium iodide exclusion assay. Briefly, 107 promastigotes from the various duration of incubated culture were washed and resuspended in PBS containing 5 µg/ml of propidium iodide (Calbiochem) and incubated at room temperature for 15 min in the dark. The stained cells were subjected to FACS (BD FACS LSRFortessa) analysis (Exλ = 488 nm; Emλ = 617 nm). 10 000 events were analyzed.

Statistical analysis

All data were expressed as the means ± SD from at least three independent experiments. Statistical analyses for all data were calculated using analysis of variance wherever applicable using Origin 6.0 software (Microcal Software). P value of less than 0.05 was considered statistically significant.

Results and discussion

Primary structure of LmPAS-PGK protein

A sequence (systematic name: LmjF.30.3380) in the L. major genome database (http/www.genedb.org/genedb/leish/) has been identified as an ORF, comprising 527 amino acid residues. It exhibits two striking features: first, its N-terminus (residues 1–115) displays limited homology to PAS domain (Figure 1) and second, the C-terminus (residues 116–527) bears ∼50% identity with PGK (Figure 1). A common PAS domain usually comprises of four/five-strand of antiparallel β-sheet and three/four flanking α-helices [7,24]. SWISS-MODEL protein modeling predicts that four antiparallel β-sheet and four flanking α-helices are conserved in this protein (Figure 2A). The C-terminus of this gene contains PGK like conserved active site residues as well as the tertiary structure (Figures 1 and 2B). These features suggested that this protein is a PAS domain-containing PGK protein (LmPAS-PGK).

Sequence alignment of LmPAS-PGK.

Figure 1.
Sequence alignment of LmPAS-PGK.

The sequence of LmPAS-PGK was aligned with the PAS domain of human Endothelial PAS domain-containing protein 1, also known as HIF-2α (EPAS1, accession number NP_001421), PAS domain of Thermotoga maritima histidine kinase (TmHK, accession number 3A0R_A), T. cruzi PAS domain-containing phosphoglycerate kinase (TcPAS-PGK, accession number ESS69068), T. brucei phosphoglycerate kinase (TbPGK, accession number AAA32120), A. deanei PAS domain-containing phosphoglycerate kinase (AdPAS-PGK, accession number EPY28995). * and red color denote active site and identical residue, respectively.

Figure 1.
Sequence alignment of LmPAS-PGK.

The sequence of LmPAS-PGK was aligned with the PAS domain of human Endothelial PAS domain-containing protein 1, also known as HIF-2α (EPAS1, accession number NP_001421), PAS domain of Thermotoga maritima histidine kinase (TmHK, accession number 3A0R_A), T. cruzi PAS domain-containing phosphoglycerate kinase (TcPAS-PGK, accession number ESS69068), T. brucei phosphoglycerate kinase (TbPGK, accession number AAA32120), A. deanei PAS domain-containing phosphoglycerate kinase (AdPAS-PGK, accession number EPY28995). * and red color denote active site and identical residue, respectively.

Biochemical characteristics of LmPAS-PGK.

Figure 2.
Biochemical characteristics of LmPAS-PGK.

(A) Based on the published X-ray crystallographic structures of the PAS domain of EPAS1 (cyan color) [54] (PDB entry code: 4xt2.2), we constructed a three-dimensional model of the PAS domain of LmPAS-PGK (green color) by knowledge-based homology modeling using the SWISS-MODEL [55] and PyMOL software [56]. (B) The ribbon structural model of the PGK domain of LmPAS-PGK was made on basis of the published X-ray crystallographic structures T. brucei PGK (PDB entry code: 13PK.1) [37]. (C) SDS–PAGE lanes: 1, lysate without IPTG (full length) 2, lysate with IPTG (full length); 3, lysate unbound flow through (full length); 4, purified full-length protein; 5, molecular weight marker; 6, (−) IPTG (Δ115); 7, (+) IPTG (Δ115); 8, flow through (Δ115); and 9, purified Δ115 protein. (D) Size-exclusion chromatography of full-length and Δ115 proteins after purification by using a Biosuite™ 250 HPLC column. (E) Change in activity of LmPAS-PGK and Δ115 LmPAS-PGK in the presence of different PAS binding ligands. (F) Activity of the LmPAS-PGK and Δ115 LmPAS-PGK in the presence of different pH values. (G) Tryptophan quenching of LmPAS-PGK under different pH conditions. (H) Velocity of LmPAS-PGK (pH 5.5) and Δ115 LmPAS-PGK (pH 7.5) with varying concentration of 3-PGA. (I) Velocity of LmPAS-PGK (pH 5.5) and Δ115 LmPAS-PGK (pH 7.5) with varying concentration of ATP.

Figure 2.
Biochemical characteristics of LmPAS-PGK.

(A) Based on the published X-ray crystallographic structures of the PAS domain of EPAS1 (cyan color) [54] (PDB entry code: 4xt2.2), we constructed a three-dimensional model of the PAS domain of LmPAS-PGK (green color) by knowledge-based homology modeling using the SWISS-MODEL [55] and PyMOL software [56]. (B) The ribbon structural model of the PGK domain of LmPAS-PGK was made on basis of the published X-ray crystallographic structures T. brucei PGK (PDB entry code: 13PK.1) [37]. (C) SDS–PAGE lanes: 1, lysate without IPTG (full length) 2, lysate with IPTG (full length); 3, lysate unbound flow through (full length); 4, purified full-length protein; 5, molecular weight marker; 6, (−) IPTG (Δ115); 7, (+) IPTG (Δ115); 8, flow through (Δ115); and 9, purified Δ115 protein. (D) Size-exclusion chromatography of full-length and Δ115 proteins after purification by using a Biosuite™ 250 HPLC column. (E) Change in activity of LmPAS-PGK and Δ115 LmPAS-PGK in the presence of different PAS binding ligands. (F) Activity of the LmPAS-PGK and Δ115 LmPAS-PGK in the presence of different pH values. (G) Tryptophan quenching of LmPAS-PGK under different pH conditions. (H) Velocity of LmPAS-PGK (pH 5.5) and Δ115 LmPAS-PGK (pH 7.5) with varying concentration of 3-PGA. (I) Velocity of LmPAS-PGK (pH 5.5) and Δ115 LmPAS-PGK (pH 7.5) with varying concentration of ATP.

Biochemical characteristics of LmPAS-PGK

To identify the biochemical characteristics of LmPAS-PGK, both the full-length protein and PAS domain deleted catalytic domain (Δ115 LmPAS-PGK, 115 amino acid deleted from the N-terminus of wild-type full-length LmPAS-PGK protein) were expressed in E. coli. Purified LmPAS-PGK and Δ115 LmPAS-PGK migrated to positions as expected from the theoretical relative molecular mass of 62 kDa (Figure 2C, lane 4) and 47 kDa protein (Figure 2C, lane 9), respectively. Figure 2D showed that both full-length and Δ115 LmPAS-PGK proteins were monomeric. For sensing environment, many of the PAS domains in sensory proteins are known to bind small molecules including heme [2527], FAD [28], FMN [29], 4-hydroxycinnamic acid [30], fatty acids [31], malate, succinate and citrate [3234]. The UV-visible spectra of LmPAS-PGK did not have any band at visible region (Supplementary Figure S1) indicating that chromophoric prosthetic groups such as heme, FAD and FMN are unlikely to function as PAS ligands in LmPAS-PGK. To identify the ligand for PAS domain, we measured the PGK activity in the presence of several well-known PAS ligands (Figure 2E). Our results suggest that the activity of LmPAS-PGK was insensitive to FAD, FMN, 4-hydroxycinnamic acid and fatty acid ligands. Although the purified enzyme did not have any heme yet its activity was stimulated by almost ∼20% in the presence of heme, indicating that the capability of heme binding to PAS domain is very weak. Although PAS-PGK sequence has no heme regulatory motif, usually containing a CP (Cys-Pro) motif [35], yet the PGK activity of the full-length enzyme was significantly enhanced by the addition of heme indicating that the association/dissociation of heme in PAS domain might regulate the catalytic activity of this enzyme.

Other PAS ligands like malate/succinate and citrate stimulated the PGK activity in both full-length and PAS-deleted proteins (Δ115 LmPAS-PGK) suggesting that this stimulation is independent of the PAS domain and is a characteristic of the PGK domain itself. It is well known that the Salmonellae PhoQ sensor kinase is directly activated when the PAS domain is exposed to pH 5.5 [36]. To determine whether LmPAS-PGK is directly activated when exposed to acidic pH, we measured PGK activity of both full-length and PAS-deleted proteins in various pH (Figure 2F). As observed in the pH curve, the activation of the full-length PGK was maximal at pH 5.5 and gradually decreased at neutral pH 7.5. On the other hand, the PGK activity of Δ115 LmPAS-PGK proteins was higher than the full-length proteins at neutral pH 7.5. Together, these results suggest that the acidic pH 5.5 relieved the autoinhibitory PAS domain from PGK catalytic domain in LmPAS-PGK. These results indicate that the PAS domain appears to have an autoinhibitory role at neutral pH 7.5 in LmPAS-PGK catalysis. The optimum activity of PAS-PGK is at around pH 5.5, close to the pH value at which histidine residues get protonated. Thus, the protonation of a histidine residue in a critical region of the PAS domain might act as the conformational switch associated with the activation. The pH-dependent fluorescence quenching (Figure 2G) suggests that acidic pH presumably triggers a conformational change in LmPAS-PGK that stimulates the catalytic activity of the PGK domain. Full-length proteins at pH 5.5 and Δ115 LmPAS-PGK proteins at pH 7.5 showed that both proteins follow standard Michaelis–Menten kinetics with respect to both of its substrates (3PGA and ATP) (Figure 2H,I). The KM and kcat values of full-length as well as Δ115 protein at both pH 5.5 and 7.5 were compared in Table 1. The KM values for ATP of the full-length protein (118 µM at pH 5.5 and 150 µM at pH 7.5) were very similar to Δ115 LmPAS-PGK proteins (110 µM at pH 5.5 and 137 µM at pH 7.5). The KM values for 3PGA of the full-length proteins (661 µM at pH 5.5 and 540 µM at pH 7.5) were comparable with Δ115 LmPAS-PGK proteins (630 µM at pH 5.5 and 520 µM at pH 7.5). These results demonstrated that its PAS domain did not affect the KM values of both the substrates. The acidic pH 5.5 significantly enhances catalysis of the full-length enzyme (3.5 s−1) compared with the PAS domain deleted enzyme (1.4 s−1), whereas the neutral pH 7.5 significantly inhibits the enzymatic activity of the full-length protein (1.3 s−1) compared with the PAS domain deleted protein (2.7 s−1). These results suggest that the N-terminal PAS domain is not directly involved in substrate binding yet it is responsible for pH-dependent activation. In the aspect of molecular mechanism of the catalysis, the enzymatic reaction of PGK briefly consists of the following events [37]. PGK exists in an ‘open’ conformation in the absence of both substrates (1,3-bisphosphoglycerate/3-phosphoglycerate and ADP/ATP). Upon binding of both the substrates, an extensive hinge-bending motion occurs, and this leads to closure of the two domains of PGK. Then, the β-phosphate of ADP initiates a nucleophilic attack on the 1-phosphate of 1,3-BPG by the help of Lys 219 (TbPGK) during the forward glycolytic reaction. The autoinhibiting PAS domain at neutral pH 7.5 may interfere with the substrate-induced domain closing, which can be the possible reason behind of lower catalytic activity.

Table 1
Catalytic activities of full-length and Δ115 LmPAS-PGK proteins

The catalytic activities were determined at 25°C as described under ‘Materials and Methods’. The values represent the mean and standard deviation for three measurements each.

Enzyme Catalytic activity parameters 
pH 5.5 pH 7.5 
KM (µM) kcat (s−1KM (µM) kcat (s−1
ATP 3PGA ATP 3PGA ATP 3PGA ATP 3PGA 
LmPAS-PGK 118 ± 4 661 ± 19 3.5 ± 0.09 3.7 ± 0.2 150 ± 5 540 ± 29 1.3 ± 0.05 1.33 ± 0.1 
Δ115 LmPAS-PGK 110 ± 7 630 ± 22 1.4 ± 0.1 1.5 ± 0.2 137 ± 7 520 ± 22 2.7 ± 0.09 2.8 ± 0.16 
Enzyme Catalytic activity parameters 
pH 5.5 pH 7.5 
KM (µM) kcat (s−1KM (µM) kcat (s−1
ATP 3PGA ATP 3PGA ATP 3PGA ATP 3PGA 
LmPAS-PGK 118 ± 4 661 ± 19 3.5 ± 0.09 3.7 ± 0.2 150 ± 5 540 ± 29 1.3 ± 0.05 1.33 ± 0.1 
Δ115 LmPAS-PGK 110 ± 7 630 ± 22 1.4 ± 0.1 1.5 ± 0.2 137 ± 7 520 ± 22 2.7 ± 0.09 2.8 ± 0.16 

Subcellular localization of LmPAS-PGK

Since the PGK activity of LmPAS-PGK is optimum at acidic pH, it raises the question of whether the mature proteins localize in acidic organelles of cells. It is well established that glycolytic enzymes are targeted to the mitochondrial matrix [38], peroxisomes [39], lysosomes [40], and flagella (or cilia) [39,41,42]. To find out the localization of the mature LmPAS-PGK protein, homogenates of RFP/CPB (N-terminal RFP tag-CPB) and FLAG-VP1 (N-terminal FLAG tag-vacuolar proton pyrophosphatase 1) overexpressing L. major cells were fractionated by differential centrifugation. Different subcellular fractions were examined by Western blotting with anti-LmPAS-PGK antibody (Figure 3A). Western blot results showed that the LmPAS-PGK protein band was recovered from both glycosomal and lysosomal fractions, where the glycosomal LmGAPDH and lysosomal CPB (as a marker protein) were concentrated (Figure 3A), suggesting that the enzyme is localized in both the glycosome and the lysosome of Leishmania. On the other hand, the LmPAS-PGK protein band was absent from the cytosol, mitochondria and acidocalcisome fractions, where the cytosolic ADK, mitochondrial APX and acidocalcisomal vacuolar proton pyrophosphatase 1 (VP1) (as a marker protein) were concentrated (Figure 3A). To confirm this observation, the full-length RFP tag LmPAS-PGK fusion in L. major promastigotes were costained with rabbit anti-LmGAPDH (glycosome-specific protein) as primary antibody and Alexa Fluor 488-conjugated anti-rabbit secondary antibody (Figure 3B). When the images were superimposed (Figure 3B), the co-localization of LmPAS-PGK with the glycosome-specific protein was observed in L. major cells. To further investigate the consequence of association of glycosomes with the acidic compartments, L. major cells were costained with DAPI (DNA-specific fluorescence dye) and LysoTracker (acidic organelle-specific fluorescence dye) and fixed on the slide (Figure 3C). When the images were superimposed (as shown in Figure 3C), the co-localization with LysoTracker was observed in L. major cells (Figure 3C). There are two acidic compartments in a Leishmania cell namely the lysosome and the acidocalcisome. Thus, it may localize in one of them or both the organelles. To examine the LmPAS-PGK localization in the lysosome, the full-length RFP tag LmCPB (lysosome-specific protein) fusion in L. major promastigotes were costained with rabbit anti-LmPAS-PGK as primary antibody and Alexa Flour 488-conjugated goat anti-rabbit antibody as secondary antibody (Figure 3D). When the images were superimposed (Figure 3D), the co-localization of LmPAS-PGK with the lysosome-specific protein was observed in L. major cells. To investigate LmPAS-PGK localization in the acidocalcisome, the full-length FLAG tag LmVP1 (acidocalcisome-specific protein) fusion in L. major promastigotes were costained with a mixture of anti-rabbit PAS-PGK antibody and anti-mice FLAG antibody as primary antibody, and a mixture of Alexa Flour 488-conjugated goat anti-rabbit antibody and Alexa Flour 546-conjugated goat anti-mice antibody as secondary antibody (Figure 3E). When the images were superimposed (Figure 3E), the co-localization of LmPAS-PGK with the acidocalcisome-specific protein was not observed in L. major cells indicating that LmPAS-PGK is absent from the acidocalcisome. To investigate LmPAS-PGK localization in the mitochondria, L. major cells were costained with DAPI and MitoTracker green (mitochondria-specific fluorescence dye) and fixed on the slide (Figure 3F). When the images were superimposed (as shown in Figure 3F), the co-localization with MitoTracker was not observed in L. major cells (Figure 3F). Since the LmPAS-PGK is absent from the acidocalcisome as well as in the mitochondria, it raises the question of whether purified organelles were intact during their isolation. To verify that we measured the organelles-specific enzymatic activity with and without 0.5% Triton X-treated purified acidocalcisome and mitochondria (Figure 3G). The enzymatic activity of Triton X-treated organelles was higher than Triton X-untreated organelles. Thus, the absence of PAS-PGK in the acidocalcisome as well as in the mitochondria is not due to breakage and release of luminal contents. Although LmPAS-PGK contains the predicted C-terminal glycosomal tri-peptide (PKL) signal sequence, yet the enzyme is present in the lysosome as well as in the glycosome. The question immediately arises as to how this protein is translocated to the lysosome. One possibility is that it may be trafficked to the lysosome from glycosome via pexophagy (fusion of glycosomes with acidic lysosomes). However, towards the C-terminal end of the PAS-PGK (before glycosomal sorting signal), there is one post-Golgi-sorting motif 504FLELL508 (Figure 1) (two reminiscent overlapping amino acid sequences: a classical dileucine base motif and tyrosine-based motif). Several groups of researcher have already shown that these types of motif in proteins are required for lysosomal translocation [4345]. Therefore, the other possibility is that this post-Golgi-sorting motif 504FLELL508 might be responsible for lysosomal localization.

Localization by Western blotting and immunofluorescence.

Figure 3.
Localization by Western blotting and immunofluorescence.

(A) Subcellular localization was performed using RFP/CPB and FLAG-VP1 overexpression cells. Lanes: 1, cell lysate; 2, cytosolic; 3, glycosomal; 4, mitochondrial; 5, lysosomal and 6, acidocalcisomal fraction. ADK, GAPDH, APX, CPB/RFP (anti-RFP antibody used) and vacuolar proton pyrophosphatase 1/FLAG (VP1) (anti-FLAG antibody used) were used in the subcellular fractions as cytosolic, glycosomal, mitochondrial, lysosomal and acidocalcisomal markers, respectively. (B) LmPAS-PGK-RFP overexpressed promastigotes were colocalized with L. major GAPDH (Alexa Flour 488 secondary antibody) (glycosomal marker). (C) LmPAS-PGK-RFP overexpressed promastigotes were colocalized with lysotracker green (acidic organelles-specific dye). (D) RFP/LmCPB (lysosomal marker) overexpressed promastigotes were colocalized with LmPAS-PGK (Alexa Flour 488 secondary antibody). (E) FLAG/LmVP1 (Alexa Fluor 546 secondary antibody) (acidocalcisomal marker) overexpressed promastigotes were not colocalized with LmPAS-PGK (Alexa Flour 488 secondary antibody). (F) LmPAS-PGK-RFP overexpressed promastigotes were colocalized with MitoTracker green (mitochondria-specific dye). (G) Measurement of the activity of marker proteins in the acidocalcisomal (AC) and mitochondrial (Mito) fractions. Pyrophosphatase and APX were used as acidocalcisomal and mitochondrial markers, respectively. Triton X was used as a membrane solublizing agent. The activity data are representative of three independent experiments.

Figure 3.
Localization by Western blotting and immunofluorescence.

(A) Subcellular localization was performed using RFP/CPB and FLAG-VP1 overexpression cells. Lanes: 1, cell lysate; 2, cytosolic; 3, glycosomal; 4, mitochondrial; 5, lysosomal and 6, acidocalcisomal fraction. ADK, GAPDH, APX, CPB/RFP (anti-RFP antibody used) and vacuolar proton pyrophosphatase 1/FLAG (VP1) (anti-FLAG antibody used) were used in the subcellular fractions as cytosolic, glycosomal, mitochondrial, lysosomal and acidocalcisomal markers, respectively. (B) LmPAS-PGK-RFP overexpressed promastigotes were colocalized with L. major GAPDH (Alexa Flour 488 secondary antibody) (glycosomal marker). (C) LmPAS-PGK-RFP overexpressed promastigotes were colocalized with lysotracker green (acidic organelles-specific dye). (D) RFP/LmCPB (lysosomal marker) overexpressed promastigotes were colocalized with LmPAS-PGK (Alexa Flour 488 secondary antibody). (E) FLAG/LmVP1 (Alexa Fluor 546 secondary antibody) (acidocalcisomal marker) overexpressed promastigotes were not colocalized with LmPAS-PGK (Alexa Flour 488 secondary antibody). (F) LmPAS-PGK-RFP overexpressed promastigotes were colocalized with MitoTracker green (mitochondria-specific dye). (G) Measurement of the activity of marker proteins in the acidocalcisomal (AC) and mitochondrial (Mito) fractions. Pyrophosphatase and APX were used as acidocalcisomal and mitochondrial markers, respectively. Triton X was used as a membrane solublizing agent. The activity data are representative of three independent experiments.

Characteristics of OE, CT, CM and KO cells

To investigate the LmPAS-PGK function in L. major, a gene replacement technique was used (Figure 4). PCR analysis with genomic DNA and Western blotting confirmed that the resulting cells no longer expressed LmPAS-PGK (Figure 4B–D). To investigate whether the growth rate of null mutants is similar to that of CT, CM or OE cells, microscopic viable cell counting analysis was performed (Figure 5A). The growth curve showed that the KO population had a slower growth rate compared with CT, CM or OE promastigotes. Flow cytometry (Figure 5B) studies suggest that KO cells had 5-fold (∼22.1%) more dead cells (PI-positive cells) than the CT (4.3%), CM (5.1%) and OE (4.0%) cells within 4 days of incubation period. These results explain why the growth rate of CT, CM or OE cells is higher than that of the KO cells. When the percentage of dead cells among KO, CT, CM and OE cells were measured at various durations of culture media, a significant amount of dead cells found in KO culture compared with CT, CM or OE cells at longer durations of culture media (Figure 5C). These results indicate that PAS-PGK may have a protecting role under unfavorable growth conditions (like nutrient stress, acidic pH or toxic metabolite). Scientists have shown that glucose starvation leads to the lowering of glycosomal pH to around pH 6.6 in T. brucei [46,47]. This slightly acidic pH may be necessary to inactivate certain proteins in glycosome for slowing down the cell growth and activate some of the essential proteins required for survival under starvation. The acidic pH sensitive LmPAS-PGK may supply ATP required by these beneficial pathways under glucose starvation and thus can help in the survivability of Leishmania. Hence, LmPAS-PGK can synthesize ATP in the glycosome as well in the acidic lysosome. To test this hypothesis, we measured the ATP concentration in cell lysate. Like LmPAS-PGK expression, the order of ATP accumulation was OE > CT = CM > KO cells (Figure 5D). Due to the impermeability of ATP in the glycosome, the ATP molecules that are consumed by hexokinase and phosphofructokinase in the upstream of the glycolytic pathway are to be regenerated inside the glycosomes through the activities of different glycosomal kinases in the downstream of the pathway. The L. major genome has six ATP producing glycosomal kinases inside the organelle i.e. PGK isoform C (LmjF.20.0100), isoform PAS-PGK (LmjF.30.3380), phosphoenolpyruvate carboxykinase (LmjF.27.1805 and LmjF.27.1810), glycerol kinase (LmjF.35.3080) and pyruvate phosphate dikinase (LmjF.11.1000). These kinase activities are essential in trypanosomes because compartmentalization of glycolysis inside the glycosomes is essential for preventing a lethal turbo-explosion of glycolysis [4850]. Our knockout studies suggest that PAS-PGK participates ∼7% of total ATP (Figure 5D). The question immediately arises as to why 93% ATP synthesis is not affected in KO cells. The possibility is that another PGK isoform C and other kinases may play an important role in glycosomal ATP synthesis in the absence of LmPAS-PGK. Next, we have measured glycolytic flux in KO cell line and compared with CT, CM and OE cell lines (Figure 5E). Since glycolytic flux is directly proportional to lactate production from glucose we have measured the concentration of lactate (the end product of glycolysis) released into the culture medium in the presence or absence of 2-deoxy d-glucose. OE cells show 20% higher lactate production than CT or CM cell lines but KO cells produce 15% lower lactate than CT or CM cell lines (Figure 5E). These data suggested that the PAS-PGK might have an important role in glycolytic flux.

Targeted gene replacement of LmPAS-PGK alleles.

Figure 4.
Targeted gene replacement of LmPAS-PGK alleles.

(A) Schematic representation of the LmPAS-PGK locus and the plasmid constructs used for gene replacement. DHFR denotes dihydrofolate reductase. (B) Agarose gel analysis of PCR-amplified products of LmPAS-PGK gene. Lane 4 shows the molecular mass marker, lanes 1 and 5, 2 and 6, and 3 and 7 correspond to PCR with genomic DNA from CT, +/− heterozygous mutants, and KO mutants, respectively, with primers external (lanes 1–3) and internal (lanes 5–7) to the LmPAS-PGK gene. The expected size of the LmPAS-PGK, NEO, and HYG gene PCR product are 3.58, 5.3 and 5.7 kb, respectively. Internal forward and reverse primers were generated from positions 345 and 1584 bp of the gene, respectively. The expected size of the PCR product is 1239 bp. (C) L. major lysate was used for Western blotting. Western blot results using rabbit anti-LmPAS-PGK and mouse anti-tubulin antibody are shown. (D) Bar diagram depicting the percentage of band intensities in the Western blot. Band intensity was quantified by ImageJ software (NIH). CM denotes complement cell lines.

Figure 4.
Targeted gene replacement of LmPAS-PGK alleles.

(A) Schematic representation of the LmPAS-PGK locus and the plasmid constructs used for gene replacement. DHFR denotes dihydrofolate reductase. (B) Agarose gel analysis of PCR-amplified products of LmPAS-PGK gene. Lane 4 shows the molecular mass marker, lanes 1 and 5, 2 and 6, and 3 and 7 correspond to PCR with genomic DNA from CT, +/− heterozygous mutants, and KO mutants, respectively, with primers external (lanes 1–3) and internal (lanes 5–7) to the LmPAS-PGK gene. The expected size of the LmPAS-PGK, NEO, and HYG gene PCR product are 3.58, 5.3 and 5.7 kb, respectively. Internal forward and reverse primers were generated from positions 345 and 1584 bp of the gene, respectively. The expected size of the PCR product is 1239 bp. (C) L. major lysate was used for Western blotting. Western blot results using rabbit anti-LmPAS-PGK and mouse anti-tubulin antibody are shown. (D) Bar diagram depicting the percentage of band intensities in the Western blot. Band intensity was quantified by ImageJ software (NIH). CM denotes complement cell lines.

Functional characterization of LmPAS-PGK.

Figure 5.
Functional characterization of LmPAS-PGK.

(A) The growth curves of CT, OE, CM (complement cell) and KO cells. (B) Cell viability of CT, OE, CM and KO cells after 4 days of incubation period as measured by flow cytometry using propidium iodide (Exλ = 488 nm, Emλ = 617 nm). (C) % of cell death among CT, OE, CM and KO cells at various time of incubation period. (D) Comparative studies of ATP content among CT, OE, CM and KO cells lysate. (E) Comparative studies of free lactate (end product of glycolysis) among CT, OE, CM and KO cells media. 2-deoxy d-glucose (2DG) used as inhibitor of glycolysis. All assays were done in three independent experiments and data represent mean ± SD. (F) The percentage of macrophages infected with CT, OE, CM and KO parasites. For each time point, 200 macrophages were counted. (G) The number of Leishmania within each infected macrophage was counted. For each time point and cell type, 200 infected macrophages were analyzed. (H) Infection in BALB/c mice. Footpad swelling of CT, OE, CM and KO were observed for the three groups (15 mice/group). (I) Parasite burden in the footpad after 6 weeks of post-infection in CT, OE, CM and KO cells. * denotes P < 0.05.

Figure 5.
Functional characterization of LmPAS-PGK.

(A) The growth curves of CT, OE, CM (complement cell) and KO cells. (B) Cell viability of CT, OE, CM and KO cells after 4 days of incubation period as measured by flow cytometry using propidium iodide (Exλ = 488 nm, Emλ = 617 nm). (C) % of cell death among CT, OE, CM and KO cells at various time of incubation period. (D) Comparative studies of ATP content among CT, OE, CM and KO cells lysate. (E) Comparative studies of free lactate (end product of glycolysis) among CT, OE, CM and KO cells media. 2-deoxy d-glucose (2DG) used as inhibitor of glycolysis. All assays were done in three independent experiments and data represent mean ± SD. (F) The percentage of macrophages infected with CT, OE, CM and KO parasites. For each time point, 200 macrophages were counted. (G) The number of Leishmania within each infected macrophage was counted. For each time point and cell type, 200 infected macrophages were analyzed. (H) Infection in BALB/c mice. Footpad swelling of CT, OE, CM and KO were observed for the three groups (15 mice/group). (I) Parasite burden in the footpad after 6 weeks of post-infection in CT, OE, CM and KO cells. * denotes P < 0.05.

Macrophages are the host cells for L. major promastigotes, therefore, we focused on the interaction of promastigotes with the macrophages. We investigated to what extent KO, CT, CM and OE cells were phagocytosed by the macrophages. The internalization rates of the KO promastigotes were similar to CT, CM and OE cells (Figure 5F). However, after phagocytosing the KO promastigotes, most of the infected macrophages did not have the KO parasites after 72 h of incubation. These data from the KO parasites suggested that the parasites were killed more easily inside the macrophages. In addition, the percentage of macrophages infected with OE increased significantly (Figure 5G). Similarly, we found that KO cells could not develop a severe disease, with an earlier onset of footpad necrosis, compared with CT or CM promastigotes (Figure 5H). These data were confirmed by the OE promastigotes, showing increased virulence in vivo infection model. The result of parasite burden during 6-week post-infection indicated that KO parasites, compared with CT or CM, had about 2-fold less parasite burden (Figure 5I) in 1 mg of footpad tissue. These findings indicated that the gene in parasites has some role in disease development in macrophages or mice.

The immediate question that comes is what mechanism is apparently involved in the disease development during infection. Recently, a group of researcher showed that human PGK1 has two additional functions other than ATP generation in cancer. One is the mitochondria-translocated PGK1 activates pyruvate dehydrogenase kinase 1 and inhibits mitochondrial pyruvate utilization to increase glycolytic production [51]. The other function is the phosphorylation of Beclin1 by PGK1 to induce autophagy [52]. Neither Leishmania gene database has Beclin1 gene nor the PAS-PGK translocates to the mitochondria. It is well known that the lysosomal dysfunction results in impaired autophagy [53]. The glycosomal and lysosomal localization of LmPAS-PGK, the regulation of glycolytic flux by this protein and protecting the role of this enzyme against old aged culture raise the question of whether this protein has any important function in autophagy. To find out the role of PAS-PGK in the induction of autophagy, we measured the degree of autophagosome formation (autophagic vacuoles) in OE, CT, CM and KO cell lines under normal and nutrient stress conditions by autophagy detection kit, RFP/ATG8 distribution, and ATG8-PE generation as the marker of autophagosome. Bright green fluorescence in the FITC filter is observed when the novel dye supplied with the autophagy detection kit (Abcam) selectively labels the autophagic vacuoles. Flow cytometry (Figure 6A) and microscopic (Figure 6B,C) studies demonstrated that the number of autophagosomes increased in KO cells compared with CT, CM or OE cells (Figure 6A–C) under both normal and nutrient stress conditions. These data were supported by RFP/ATG8 overexpression system, where the expression of N-terminally RFP-fused ATG8 in L. major facilitated the monitoring of autophagy by using fluorescence microscopy to detect the presence of RFP-labelled puncta (a marker for tracking autophagosome formation). KO cells showed that the percentage of parasites with puncta and the number of puncta per cell increased with respect to CT, CM or OE cells in mid-logarithmic phase of growth (Figure 6D,E). Autophagosomes of L. major cells are frequently quantified by the amount of conversion of ATG8 to membrane-bound ATG8-PE. Conversion of RFP/ATG8 to membrane-bound RFP/ATG8-PE in KO cells was confirmed through immunoblot with anti-RFP antibody. The expression of RFP/-ATG8-PE in KO cells was ∼3-fold higher than CT or CM cells whereas OE parasites showed lower expression of ATG8-PE when compared with CT or CM parasites (Figure 6F). Bafilomycin A1 and wortmannin, two well-known inhibitors of autophagy, were used as control while studying the distribution of RFP/ATG-PE. Bafilomycin A1 inhibits the fusion of autophagosome and lysosome leading to the accumulation of autophagosomes whereas wortmannin prevents the autophagosome formation. Similarly, bafilomycin A1-treated cells showed higher amount of RFP/ATG-PE expression whereas wortmannin prevents the RFP/ATG-PE expression in all type of cell lines. Altogether, these data suggest that the LmPAS-PGK can regulate autophagosome formation in Leishmania promastigotes.

Role of LmPAS-PGK in autophagy.

Figure 6.
Role of LmPAS-PGK in autophagy.

(A) Autophagic flux measurement of the CT, OE, CM and KO cells under normal and stressed conditions by flow cytometry using autophagy detection kit (Exλ = 488 nm, Emλ = 517 nm). St denotes stressed conditions (glucose starvation). (B) The same samples as (A) were visualized by fluorescence microscopy. (C) The same images as (B) in brightfield background. (D) Formation of RFP/ATG8 puncta in CT, OE, CM and KO cells under normal and stressed conditions using fluorescence microscope. (E) The same images as (D) observed in brightfield background. (F) Western blot analysis of RFP/ATG8 using anti-RFP antibody detecting both RFP/ATG8 and RFP/ATG8-PE. Wortmannin and bafilomycin A1 were used as controls. Bafilomycin A1 (inhibitor of the late phase of autophagy) and glucose starvation induced autophagosome formation in parasites was also confirmed by the increased expression of the ATG8-PE. Inhibitor of early phase of autophagy, wortmannin, was found to inhibit the autophagosome formation in KO parasites.

Figure 6.
Role of LmPAS-PGK in autophagy.

(A) Autophagic flux measurement of the CT, OE, CM and KO cells under normal and stressed conditions by flow cytometry using autophagy detection kit (Exλ = 488 nm, Emλ = 517 nm). St denotes stressed conditions (glucose starvation). (B) The same samples as (A) were visualized by fluorescence microscopy. (C) The same images as (B) in brightfield background. (D) Formation of RFP/ATG8 puncta in CT, OE, CM and KO cells under normal and stressed conditions using fluorescence microscope. (E) The same images as (D) observed in brightfield background. (F) Western blot analysis of RFP/ATG8 using anti-RFP antibody detecting both RFP/ATG8 and RFP/ATG8-PE. Wortmannin and bafilomycin A1 were used as controls. Bafilomycin A1 (inhibitor of the late phase of autophagy) and glucose starvation induced autophagosome formation in parasites was also confirmed by the increased expression of the ATG8-PE. Inhibitor of early phase of autophagy, wortmannin, was found to inhibit the autophagosome formation in KO parasites.

Conclusion

These data unequivocally demonstrate that PAS-PGK act as an acidic PGK. On the basis of physiological function, PAS-PGK proteins constitute a previously unknown class of PAS-containing protein that differ significantly from the known PAS-containing protein sensors like histidine and serine/threonine kinases, chemoreceptors and photoreceptors, clock proteins, voltage-activated ion channels, cyclic nucleotide phosphodiesterases, regulators of hypoxia responses, and modulators of embryological development, etc. [7]. Our results suggest that the N-terminal domains of PAS-PGK act as a regulator for showing optimum PGK activity at acidic pH. The acidic pH presumably triggers a conformational change that relieves the PAS domain induced inhibition from the C-terminal catalytic PGK domain, resulting in the synthesis of the optimum level of ATP, which then associates with the lysosomal function and cell survival. The unusual presence of the PAS domain on the N-terminal of the LmPAS-PGK makes it unique from all the other known PGKs.

Abbreviations

     
  • ADK

    adenosine kinase

  •  
  • AP

    alkaline phosphatase

  •  
  • APX

    ascorbate peroxidase

  •  
  • CLF

    crude lysosomal fraction

  •  
  • CM

    complement cell

  •  
  • CPB

    cysteine peptidase B

  •  
  • CT

    control cell

  •  
  • GAPDH

    glyceraldehyde 3 phosphate dehydrogenase

  •  
  • IPTG

    isopropyl β-d-1-thiogalactopyranoside

  •  
  • KO

    LmPAS-PGK knockout cell

  •  
  • LmPAS-PGK

    PAS domain-containing phosphoglycerate kinase from Leishmania major

  •  
  • OE

    LmPAS-PGK overexpressing cell

  •  
  • PAS

    Per (Drosophila period clock protein)-Arnt (vertebrate aryl hydrocarbon receptor nuclear translocator)-Sim (Drosophila single-minded protein)

  •  
  • PGK

    phosphoglycerate kinase

Author Contribution

S.A. and A.A. designed research; A.A., S.B., S.D. and A.M. performed research; S.A. analyzed data and wrote the paper.

Funding

This work was supported by Department of Science and Technology (EMR/2016/001415), CSIR fellowships (to A.M. and S.B.), and University Grants Commission fellowships (to A.A. and S.D).

Acknowledgements

We thank Dr. S. M. Beverley for providing pXG-B2863, pXG-Neo, and pXG-Hyg vectors; Prof. Emmanuel Tetaud (Université de Bordeaux) for providing pNUS-mRFPnD vector; and Dr. A. K. Datta for Leishmania donovani adenosine kinase antibody.

Competing Interests

The Authors declare that there are no competing interests associated with the manuscript.

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