Biochemical and inhibition studies of glutamine synthetase from Leishmania donovani

Accepted Manuscript Biochemical and inhibition studies of glutamine synthetase from Leishmania donovani Vinay Kumar, Shailendra Yadav, Neelagiri Soumya, Rohit Kumar, Neerupudi Kishore Babu, Sushma Singh PII:

S0882-4010(16)30773-2

DOI:

10.1016/j.micpath.2017.03.024

Reference:

YMPAT 2179

To appear in:

Microbial Pathogenesis

Received Date: 10 November 2016 Revised Date:

21 March 2017

Accepted Date: 23 March 2017

Please cite this article as: Kumar V, Yadav S, Soumya N, Kumar R, Babu NK, Singh S, Biochemical and inhibition studies of glutamine synthetase from Leishmania donovani, Microbial Pathogenesis (2017), doi: 10.1016/j.micpath.2017.03.024. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT 1

Biochemical and inhibition studies of glutamine synthetase from Leishmania donovani

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Vinay Kumar, Shailendra Yadav, Neelagiri Soumya, Rohit Kumar, Neerupudi Kishore Babu,

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Sushma Singh*

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Department of Biotechnology, National Institute of Pharmaceutical Education and Research,

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SAS Nagar, Mohali-160062, Punjab, India.

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* Corresponding author at Department of Biotechnology, National Institute of

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Pharmaceutical Education and Research, SAS Nagar, Mohali-160062, Punjab, India.

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Tel.: +91-172-2292208; fax: +91-172-2214692.

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Running title: Glutamine synthetase from Leishmania donovani

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E-mail : [email protected] (Dr.Sushma Singh).

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Abstract

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Leishmaniasis is a group of tropical diseases caused by protozoan parasites of the genus

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Leishmania. Leishmania donovani is a protozoan parasite that causes visceral leishmaniasis, a

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fatal disease if left untreated. Chemotherapy for leishmaniasis is problematic as the available

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drugs are toxic, costly and shows drug resistance, hence, there is a necessity to look out for

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the novel drug targets, chemical entities and vaccine. Glutamine synthetase (GS) catalyzes

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the synthesis of glutamine from glutamate and ammonia. In the present study, we have

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identified and characterized GS from L. donovani. The nucleotide sequence encoding

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putative glutamine synthetase like sequence from L. donovani (LdGS, LDBPK_060370) was

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cloned. A 43.5 kDa protein with 6X-His tag at the C-terminal end was obtained by

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overexpression of LdGS in Escherichia coli BL21 (DE3) strain. Expression of native LdGS in

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promastigotes and recombinant L. donovani glutamine synthetase (rLdGS) was confirmed by

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western blot analysis. An increase in expression of GS was observed at different phases of

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growth of the parasite. Expression of LdGS in promastigote and amastigote was confirmed by

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western blot analysis. Immunofluorescence studies of both the promastigote and amastigote

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stages of the parasite revealed the presence of LdGS in cytoplasm.. GS exists as a single copy

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gene in parasite genome. Kinetic analysis of GS enzyme revealed Km value of 26.3 ± 0.4 mM

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for L- Glutamate and Vmax value of 2.15 ± 0.07 U.mg-1. Present study confirms the presence

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of glutamine synthetase in L. donovani and provides comprehensive overview of LdGS for

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further validating it as a potential drug target.

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Key words: glutamine synthetase; Leishmania; kinetics; localization

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1. Introduction

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Leishmaniasis is a group of vector-borne disease caused by an intracellular obligate

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protozoan parasite belonging to the genus Leishmania. Clinical forms of leishmaniasis

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include cutaneous, mucosal and visceral leishmaniasis. Visceral leishmaniasis (VL) also

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known as kala-azar is fatal in over 95 % of cases if left untreated. Around half a million new

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cases of VL are reported worldwide each year and over 90 % of new cases occur in

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Bangladesh, Brazil, Ethiopia, India, South Sudan and Sudan [1–3]. VL is caused by two

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leishmanial species, Leishmania donovani or Leishmania infantum, depending on the

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geographical area. L. infantum infects mostly children and immune suppressed individuals,

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whereas L. donovani infects all age groups [1]. Infection to the mammalian host is caused by

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flagellated metacyclic promastigotes that are deposited in the skin during feeding of the sand

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fly vector. Current treatment regimen for the disease includes pentavalent antimonials,

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amphotericin B, paromomycin and miltefosine [4]. Antileishmanial drug treatment is

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problematic as the available drugs are toxic, costly and face drug resistance particularly in

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India [5,6]. Increasing resistance of Leishmania towards pentavalent antimonials has raised

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serious concern. Therefore, there is an urgent need for efficacious drugs and vaccines against

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VL. Understanding of the various metabolic pathways and the essentiality of enzymes

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involved in them will provide more information regarding the potential of metabolic proteins

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to be used as drug target against leishmaniasis. One such enzyme under consideration is

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glutamine synthetase. Glutamine synthetase (GS; EC 6.3.1.2, also known as γ-glutamyl:

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ammonia ligase) catalyzes the ATP dependent synthesis of glutamine from glutamate and

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ammonia (Fig. 1) [7].

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Fig. 1

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In the first step of the enzymatic reaction, glutamate is phosphorylated to yield gamma

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glutamyl phosphate. Ammonia then replaces the phosphate to form the amide moiety of

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glutamine. This catalytic mechanism is considered to be same for all the existing forms of the

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GS as is evident by the presence of highly conserved residues at the glutamate and ammonia 3

ACCEPTED MANUSCRIPT binding sites [8,9]. In many organisms ammonia assimilation by GS leads to synthesis of

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glutamate from glutamine by the enzyme GS. This pathway is referred to as GS/GOGAT

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pathway [10]. Evolution of GS, one of the most ancient genes is considered a good molecular

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clock [11,12]. It is a ubiquitous enzyme, which is involved in nitrogen metabolism, recycling

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of neurotransmitter glutamate, and the synthesis of glutamine for the production of amino

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acids, sugars and glucosamine-6-phosphate [13]. Amino acids are essential components of

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metabolism in kinetoplastids. Some amino acids in Leishmania and Trypanosoma are

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reported to act as source of energy production and as triggers to differentiation process [14–

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16]. Amino acids can be used as carbon and energy sources and they act as crucial nutrients

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during the Trypanosoma cruzi life cycle. They also participate in several biological processes

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that help the parasite adjust to various environmental changes [17,18]. Among other amino

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acids, glutamate is required for the metacyclogenesis of T. cruzi [19,20]. Moreover,

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glutamate is converted to glutamine, the amino donor for several essential metabolic

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pathways in Leishmania, including pyrimidine and amino sugar synthesis [21,22]. L-proline

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is the main source of energy generation for Leishmania promastigotes [23]. Till date, three

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distinct forms of GS have been reported on the basis of their length of amino acids: GSI,

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consisting of 12 identical subunits (450 to 470 amino acids each), is present mostly in

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prokaryotes but, have been recently identified in mammals and plants also [24,25]. The

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quaternary structure of GSII enzymes has been controversial, however the octameric as well

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as decameric arrangement of GSII subunits (350 to 420 amino acids each) is primarily found

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in eukaryotes and some bacteria (Rhizobiaceae and Streptomycetaceae families, which also

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have GSI, and GSIII) [26,27]. As GSI, GSIII is composed of 12 identical subunits (but with

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about 700 amino acids each), and was first found in Bacteroides fragilis and identified later

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in a few more anaerobic bacteria and cyanobacteria [28–30].

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In Leishmania chagasi GS was identified as T cell antigen [31]. It is expressed by the

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amastigote stage and is reported to stimulate immune T- cell proliferation and cause increase

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in IFN-γ levels. Thus, GS acts as a potential target for the immune response during

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mammalian infection [31]. It was reported in L. mexicana, that inhibition of either TCA cycle

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or GS strongly inhibits amastigote growth and viability in vitro and in infected macrophages

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[21]. There are several essential enzymes in L. donovani which utilize glutamine as their

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substrate, one of them being L. donovani, asparagine synthetase (AS). There are two

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structurally distinct types of AS: A and B. AS-A from Trypanosoma brucei, T. cruzi and L.

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ACCEPTED MANUSCRIPT donovani parasites were reported not only to use ammonia but also glutamine as nitrogen

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donor [32–34].

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The internal L-Glutamine pool acts as a sensor of external nitrogen. In Salmonella enterica

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serovar Typhimurium, mutation of glnA resulted in a marked reduction of virulence and a

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survive ability to host cells [35,36]. Streptococcus suis serotype 2 (S. suis2) is an important

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pathogen, responsible for diverse diseases in swine and human [37,38]. Deletion of GS gene

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(∆glnA mutant) had significantly reduced the virulence of S. suis2, suggesting that glutamine

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metabolism is especially important for the virulence of pathogens [39]. In Mycobacterium

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tuberculosis, the glnA-1 gene encodes a class I GS (GSI) enzyme that is released into the

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culture medium and plays a crucial role in pathogenicity [40]. Also glnA-1 mutant strain of

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Mycobacterium bovis infects THP-1 cells with reduced efficiency and also exhibited

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attenuated growth in macrophages [41]. Bacillus subtilis GS was reported to be moonlighting

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protein which, apart from its enzymatic functions, has a critical role in the control of gene

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expression [42].

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GS inhibitors have also been reported to inhibit mycobacterial and L. mexicana glutamine

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synthetase [21]. Because of its importance, the enzyme has been projected as a target for

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antimicrobial drugs. GS has been identified as a potential drug target in silico approach in

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Plasmodium falciparum and M. tuberculosis [43,44]. Amino acids participate in a variety of

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metabolic pathways leading to the synthesis of products which are crucial for survival of

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parasites. Therefore, metabolic enzymes can be considered good target for drug design. GS

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has an important role in the metabolic process of glutamate. To our knowledge, little is

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known about the biological function of GS in L. donovani. This paper is the first to report the

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cloning and characterization of the glutamine synthetase from L. donovani. Present study

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would be helpful in ascertaining functional aspects of the enzyme in the parasite.

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2. Materials and methods

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2.1. Materials

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All the restriction enzymes were obtained from Bangalore Genei Pvt. Ltd., India. L-Glutamic

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acid, GenEluteTM plasmid miniprep kit, GenEluteTM gel extraction kit, oligonucleotide

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primers, Trizol reagent, sodium bicarbonate, streptomycin, penicillin G, His-Select® HF

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nickel affinity gel and alkaline phosphatase conjugated anti-rabbit IgG were purchased from

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Sigma (St. Louis, USA). Taq DNA polymerase, dNTPs (deoxynucleotide triphosphates) were

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from Invitrogen (Carlsbad, USA), T4 DNA ligase (NEB Pvt. Ltd., UK),Wizard gDNA

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ACCEPTED MANUSCRIPT purification kit was purchased from Promega Biotech India Pvt. Ltd. PCR DIG Probe

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Synthesis kit and DIG immunological detection kit (Roche), Anti-His monoclonal antibody

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was purchased from Calbiochem. Poly-L-lysine coated coverslips was from BD Biosciences

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and Polyclonal anti-rabbit GS antibody was customized from Abgenex Pvt. Ltd.,

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Bhubaneswar, India. RPMI-1640 HEPES-modified medium and foetal bovine serum were

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purchased from Gibco/BRL (Life Technologies Scotland, UK). Other chemicals used in this

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study were of analytical grade and commercially available.

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2.2. Parasite and culture conditions

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L. donovani (wild type, MHOM/80/IN/Dd8) promastigotes were cultured and maintained at

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24 °C in RPMI 1640 HEPES-modified medium supplemented with 0.2 % sodium

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bicarbonate, 100 µg/mL penicillin G, 100 µg/mL streptomycin, 100 µg/mL gentamicin and

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10 % heat inactivated foetal bovine serum (FBS). Medium was maintained at pH 7.2. For the

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generation of axenic amastigotes, 1x 108 cells of L. donovani wild type promastigotes were

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grown in potassium buffered RPMI-1640 base medium (pH 5.5) at 37 °C with 5 % CO2

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according to the method described by Debrabant, et al. [45] and used after two to three

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passages.

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2.3. Southern blot analysis

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Genomic DNA was isolated from L. donovani promastigotes using Wizard gDNA

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purification kit (Promega Biotech India Pvt. Ltd). Approximately 5 µg of genomic DNA was

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digested with selected restriction endonucleases (internal and external cutters of the gene),

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and subjected to electrophoresis in 0.7 % agarose gels and, then transferred to a positively

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charged nylon membrane (mdi, Advanced Microdevices Pvt. Ltd). DIG-labelled full length

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LdGS

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5'-GGAATTCCATATGTCCTCGTCAAATAAGCAGACC-3'

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5'-CCCAAGCTTGAAAGCATTAC GCATCCAGTCC-3’(antisense) primer with the PCR

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DIG Probe synthesis kit (Roche). The resulting digoxigenin (DIG)-labelled products was

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used as probe. The membrane was hybridized overnight at 42 °C with the DIG-labelled LdGS

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probe (10 µL of PCR product). Following hybridization, membranes were washed twice

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under low-stringency conditions (2X SSC with 0.1 % sodium dodecyl sulfate for 5 min at

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25 °C and twice under high-stringency conditions (0.5X SSC with 0.1 % SDS for 15 min at

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68 °C, where 1X SSC comprises 150 mM sodium chloride and 15 mM sodium citrate

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probe

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using and

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(pH 7.0). Finally, blot was developed with DIG immunological detection kit (Roche)

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according to the manufacturer’s instructions.

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2.4. Cloning of L. donovani glutamine synthetase and sequence analysis

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The genomic DNA was isolated from Wizard gDNA purification kit procured from Promega

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Biotech India Pvt. Ltd. by following manufacturer’s protocol and used as template to amplify

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GS. Primers were designed based on the putative sequence of L. donovani glutamine

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synthetase

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5'-GGAATTCCATATGTCCTCGTCAAATAAGCAGACC-3' and the antisense primer with

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a flanking HindIII site 5'-CCCAAGCTTGAAAGCATTACGCATCCAGTCC-3' were

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synthesised. The PCR was performed in a 50 µL reaction consisting of 1X PCR buffer, 2 mM

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MgCl2, 400 µM of dNTPs, 0.4 µM of each primer, 250 ng of genomic DNA and 1U of Taq

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DNA polymerase enzyme (Invitrogen).The conditions used to amplify the GS gene was hot

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start at

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annealing at 58.9 °C for 1 min, elongation at 72 °C for 1 min and final extension of 10 min at

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72 °C. The PCR product was cloned into pET30a (Novagen), to produce the recombinant

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pET30a-LdGS plasmid which was further confirmed by DNA sequencing (1st BASE, Axil

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Scientific Pvt Ltd). Determination of molecular weight and isoelectric point of the LdGS was

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performed with the ExPasy program (http://web.expasy.org/protparam/). Multiple sequence

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alignment

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(http://www.ebi.ac.uk/Tools/msa/clustalo/)

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(http://www.ch.embnet.org/software/BOX_form.html),

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mitochondrial targeting sequence in LdGS protein (AMQ34913.1) was further predicted by

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MitoProt software (https://ihg.gsf.de/ihg/mitoprot.html).

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2.5. Expression, purification of recombinant LdGS and antibody production

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The positive clone was transformed in to E. coli BL21 (DE3) strain. Protein expression was

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induced with 0.1 mM IPTG, for 14 h at 25 °C when the A600 reached between 0.4 and 0.6.

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The cells were harvested by centrifugation at 4 °C, 6000g and resuspended in 20 mM Tris-

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HCl buffer (pH 7.8) and incubated on rocking platform for 30 min at 4 °C after the addition

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of lysozyme (100 µg/mL) and 0.1 % Triton X-100 to cell suspension. The resulting cell

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suspension was lysed by sonication for 10 short bursts of 30 sec with 30 sec interval cooling

a

sense

primer

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NdeI

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(underlined)

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performed

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Clustal

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program version

Presence

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95 °C for 5 min, followed by 30 cycles of denaturation at 94 °C for 1 min,

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ACCEPTED MANUSCRIPT on ice using Hielscher sonicator set at 80 % amplitude. The supernatant obtained as a soluble

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fraction after centrifugation at 12,000g for 30 min at 4 °C was loaded onto HIS-Select® HF

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nickel affinity column which was pre equilibrated with 20 mM TrisCl buffer (pH 7.8), 100

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mM NaCl and 10 mM imidazole and 0.1 % Triton X-100. The column was washed with

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buffer containing (20 mM Tris-HCl, pH 7.8; 10 mM and 20 mM imidazole; 300 mM sodium

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chloride and 0.1 % Triton X-100). Finally, bound protein was eluted with elution buffer

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containing (20 mM Tris-HCl, 150 mM imidazole and 300 mM NaCl). The purified rLdGS

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protein was dialyzed overnight against 20 mM Tris-HCl (pH 7.8) at 4 °C. Protein estimation

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was done by Bicinchoninic acid (BCA) method using bovine serum albumin (BSA) as the

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standard and its purity was determined on 10 % SDS-PAGE. Purified rLdGS protein was

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resolved on 10% SDS-PAGE and stained with coomassie brilliant blue dye. A 43.5 kDa

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protein band was excised and given for customised production of polyclonal antibody in

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rabbit (Abgenex Pvt. Ltd., Bhubaneswar India).

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2.6. Crude lysate preparation of L. donovani promastigotes and amastigotes

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1x108 L. donovani promastigotes were harvested by centrifugation at 6000g, washed three

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times with phosphate buffer saline (pH 7.4). The cell pellet was resuspended in lysis buffer

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(20 mM Tris pH 7.8, 0.5 µg/mL aprotinin, 1 mM PMSF and 0.5 µg/mL leupeptin) and

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incubated on ice for 10 min before lysis by freeze thaw. Amastigotes were pelleted by

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centrifugation at 3000g and resuspended in lysis buffer containing 20 mM Tris (pH 7.8),

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0.5 % (v/v) Triton X-100,10 µg/ml leupeptin, 50 µg/ml aprotinin and incubated on ice for 30

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min. The cells were lysed by repeated freeze-thaw cycles for 15 sec in liquid nitrogen and 30

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sec at 37 °C, for 8 times. The supernatant was collected by centrifugation at 12,000g for 30

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min at 4 °C and used for western blot analysis and determination of specific activity of LdGS

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in promastigotes.

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2.7. Protein immunoblotting

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For western blotting purified rLdGS protein samples were resolved on SDS-PAGE and

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transferred onto nitrocellulose membrane using electrophoresis transfer cell (BioRad). The

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membrane was blocked with 5 % BSA in 1X TBST. After washing, the membrane was

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incubated with monoclonal anti-His primary antibody (Calbiochem, 1:5000) followed by

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anti-mouse IgG alkaline phosphatase conjugate as secondary antibody (Sigma, 1:10000). 8

ACCEPTED MANUSCRIPT Also to check the expression of GS in promastigotes and amastigotes, cell lysate was

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prepared and protein concentration in total cell lysate was determined by Bradford method

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[46]. Approximately 100 µg of protein from each cell lysate was resolved by SDS-PAGE.

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Proteins were transferred to nitro-cellulose membrane and processed for western blot analysis

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with anti-LdGS primary antibody at 1:2500 dilutions and anti-rabbit IgG alkaline phosphatase

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conjugate was used as secondary antibody (Sigma, 1:5000). The respective protein bands

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were visualized by incubating with nitro blue tetrazolium (NBT) and 5-bromo-4-chloro3-

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indolyl phosphate disodium salt (BCIP) as substrates.

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2.8. Immunolocalization of GS

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Localization of GS in L. donovani was studied by immunolocalization as previously

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described [47]. Briefly, L. donovani promastigote and amastigote cells were fixed (4 %

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paraformaldehyde) and

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permeabilized (0.1 % v/v Triton X-100 in PBS for promastigote and 0.2 % v/v for

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promastigote) for 10 min. Blocking was done using blocking buffer (0.1 % BSA in PBS

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containing 0.1 % Triton X-100). The cells were incubated with rabbit anti-LdGS antibody

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(1:100) for 90 min as primary antibody followed by Alexa Fluor 488 anti-rabbit IgG

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conjugate (1:100, Invitrogen) as secondary antibody for 1h in the dark condition. DAPI

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(10 µg/mL in PBS) was used to stain the nuclear and kinetoplast DNA. Unbound antibody at

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each step was removed by washing with PBS, five times for five min. Finally, oil immersed

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slides were observed under 100X magnification using a Nikon Eclipse E600 epi-fluorescence

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microscope.

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2.9. Recombinant LdGS enzyme assay

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rLdGS activity was determined using a colorimetric method described by Gawronski and

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Benson [48]. 100 µL of assay mixture contained 10 mM Tris–HCl buffer (pH 7.8), 5 mM

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NH4Cl, 5 mM MgCl2, 20 mM L-glutamate, 3 mM ATP, and purified rLdGS. The reaction

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mixture was incubated at 37 °C for 20 min at 150 rpm. To detect phosphate released, the

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following solutions were used: reagent A, 12 % w/v L-ascorbic acid in 1 N HCl; reagent B,

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1.5 % w/v ammonium molybdate tetrahydrate in H2O; reagent C consisting of a mixture of

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two parts A and one part B; reagent D, 2 % sodium citrate tribasic dehydrate in 2 % acetic

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acid. Standard curve was prepared by using potassium phosphate. Reagent C was prepared

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ACCEPTED MANUSCRIPT immediately prior to use as a color developmental reagent (75 µL/well), its low pH

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terminated the reaction. Reagent D (75 µL/well) was added to the wells to stop colour

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development. The activity of rLdGS was measured by calculating the amount of phosphate

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released in the enzyme assay mixture, which was estimated by the change in absorbance at

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655 nm using microplate reader (BioTek, USA). The inhibitors (L-methionine sulfoximine,

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L-methionine sulfone and Phosphinothricin) were screened for their ability to inhibit the

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catalytic efficiency of recombinant LdGS. All reaction components including inhibitor 10-

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100 µM were incubated for 5 min at 37 oC. Reaction mixture without inhibitor and solvent

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for inhibitor (water), served as the control. After incubation, substrate (glutamate) was added

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and all steps were followed according to protocol for rLdGS enzyme activity.

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2.9.1. Effect of pH and divalent metal ion and temperature on rLdGS

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To determine the optimum pH of the rLdGS, buffers with different pH values were used in

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the assay mixture. Imidazole buffer was used in the range of pH 6-7, Tris–HCl buffer was

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used in the range of pH 8-9 and sodium acetate buffer was used in the pH range of 10-11.

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Effect of different metal ions (Mn2+, Mg2+, Ca2+, Zn2+ and Ni2+) on rLdGS activity was

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studied by incubating the enzyme with 10 mM concentration of different metal chlorides for

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20 min at 37 °C. The effect of temperature on rLdGS was determined in Tris-HCl buffer at

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different temperatures (5-70 °C).

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2.9.2. Determination of kinetic parameters

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Kinetic parameters of the purified rLdGS were determined for the substrate, L-glutamate

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(concentration range of 0 to 40 mM), ammonium chloride, and ATP (concentration range of 0

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to 10 mM), in the forward reaction. The initial velocity (Vo) was measured over the range of

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L-glutamate, concentrations from 5 to 40 mM. . In this assay system, all kinetic

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measurements were performed at 37 °C. All data were corrected by eliminating extra

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phosphate, including non-enzymatic ATP hydrolysis in the control mixture from which L-

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glutamate was omitted. Kinetic parameters were determined from velocity vs. substrate

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curves by fitting the data to the Lineweaver-Burk plot.

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2.10. In vitro antileishmanial assay

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ACCEPTED MANUSCRIPT In vitro leishmanicidal activity of various inhibitors (L-methionine sulfoximine,

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L-methionine sulfone, and Phosphinothricin) were investigated on L. donovani promastigotes

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with MTT assay [49]. 2 x105 cells/200 µL/well were seeded in 96 well plate and kept for

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incubation at 24 °C. After 48 h, different dilutions of each inhibitor were prepared and added

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to the cells in triplicates and further kept for incubation at 24 °C for 48 h. MTT was added at

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a final concentration of 400 µg/mL and further incubated at 37 °C for 4 h. The cells were

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centrifuged at 3000g for 10 min and the supernatant was removed. The resultant purple

317

formazan formed was dissolved in 100 µL DMSO and finally absorbance was read at 540 nm

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on a Tecan microplate reader. The IC50 values of the treated leishmanial cells were calculated

319

relative to the untreated control cells and the results were expressed as the inhibitor

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concentration at which there was 50 % inhibition of the parasite viability.

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3. Results

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3.1. In silico analysis of L. donovani glutamine synthetase

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Genomic DNA from L. donovani wild type promastigotes was used as a template to amplify

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the full length 1137 bp GS by PCR and product obtained was further cloned and the sequence

325

was confirmed by automated sequencing. The nucleotide sequence was submitted to

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GenBank under the Accession No. KT907048. The ORF encoded for a putative polypeptide

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of 379 amino acids with an expected molecular weight of 43.5 kDa. Multiple sequence

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analysis revealed 41 % identity between H. sapiens GS (NCBI, NP_001028216) and L.

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donovani GS (NCBI, AMQ34913) sequences. Also L. donovani GS protein sequence was

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found to be 84.7 % identical to L. mexicana (NCBI, AAY45994), 62.1 % identical to T. cruzi

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(NCBI, XP_820989), 60.9 % identical to T. brucei (NCBI, XP_846120), 41.6 % identical to

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S. cerevisiae (NCBI, EDN61170), 41.2 % identical to C. familiaris (NCBI, AAN41001), 37.9

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% identical to Z. mays (NCBI, AAM00242), 17.8 % identical to M. tuberculosis (NCBI,

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AAB70038) and 19.8 % identical to E. coli (Fig 2A, Table.1).

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Fig. 2A

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Table. 1

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Amino acid residues involved in binding of glutamate (E134, E136, N248, G249, H253, R299, R319, E338, R340), ATP (G187, S257, R324), and ammonia (D63, S65, E196, E203, E305) are conserved in all organisms. Amino acid position indicated corresponds to human GS [26]. A sequence identity tree was constructed (Fig.2B) using the L. donovani GS amino acid sequence and representative GS sequences from other organisms.

346 347

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Fig. 2B

348

The tree indicates close evolutionary relationship of L. donovani and L. mexicana. Among the

349

kinetoplastid protozoa GS sequences are closer to T. cruzi in sequence identity analysis but

350

are distantly apart from H. sapiens, S. cerevisiae and Z. mays.

351

3.2. Genomic organisation of L. donovani GS gene by southern blot analysis

352

In order to determine the copy number of the L. donovani GS gene, southern blot was

353

performed as described under materials and methods using 1140 bp DIG labelled PCR 13

ACCEPTED MANUSCRIPT product as a probe. The genomic DNA from L. donovani promastigotes was digested with the

355

following restriction enzymes: EcoRI, SalI, NcoI, XhoI and XmaI which have no internal sites

356

within L. donovani GS gene; and NsiI which have one internal site as shown in Fig. 3A.

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Fig. 3 A, B

358

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Digestion with enzymes having no internal site resulted in single intense band while with

360

enzyme (NsiI) having one internal site resulted in two bands. . The observed restriction

361

digestion pattern suggests that L. donovani GS gene exists as single copy number gene (Fig.

362

3B). Sequence analysis of the parasite genome also reveals the presence of only one copy of

363

the GS gene.

364

3.3. Expression and purification of full length rLdGS enzyme

365

The 1137 bp LdGS gene was cloned in frame into pET30a vector with its own start codon but

366

without stop codon to enable expression of C-terminal His tag. The protein overexpression

367

was carried out as described under materials and methods. The LdGS protein was

368

overexpressed as soluble, active protein in BL21 (DE3) strain of E. coli. The LdGS protein

369

with a molecular weight of 43.5 kDa that matched with the estimated molecular weight

370

(according to amino acid composition with 6-His residues) was induced (Fig. 4A). The

371

rLdGS was purified by nickel affinity chromatography column. Different elution fractions of

372

purified rLdGS were analyzed by 10 % SDS–PAGE under denaturing conditions. The rLdGS

373

protein obtained after purification showed only single band of 43.5 kDa (fused with histidine

374

tag) on the SDS–polyacrylamide gel stained with coomassie brilliant blue R250 (Fig. 4B).

375

Yield of the purified protein was found to be approximately 8 mg/L of induced bacterial

376

culture. The enzyme was stable up to three days with no apparent change in activity (data not

377

shown).

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378 Fig. 4. A, B

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3.4. Western blot analysis of LdGS

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Different concentrations of purified rLdGS were analyzed by 10 % SDS–PAGE. Expression

382

of rLdGS was confirmed further by western blot analysis using anti-His antibody (Fig. 4C)

383

and expression of GS in promastigote and amastigote was confirmed by anti-LdGS antibody

384

which revealed the band of expected 43.5 kDa size (Fig. 4D). Further, western blot was also

385

performed using promastigote cell lysate (50 µg) at different time intervals of parasite

386

growth. The anti-LdGS antibody detected the band of expected size at all the time points

387

which clearly demonstrates that the enzyme is expressed in different phases of growth of the

388

parasite (Fig. 4E).

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Fig. 4. C-E

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3.5. Intracellular localization of LdGS

392 393 394 395 396 397 398 399 400

The subcellular localization of LdGS was studied by immunofluorescence microscopy in both the promastigotes and amastigote stages of the parasite. In both the cases a homogenous diffused green fluorescence pattern throughout the cytosol was observed as shown in overlay image (panel (iv) of Fig. 5 A, B) whereas there was no co-localization with the DAPI stain. Also there was no green fluorescence observed in case of promastigotes and amastigotes fixed with pre-immune sera which served as negative control (data not shown). Our results demonstrate that LdGS is a cytosolic protein however the possibility of mitochondrial localization should not be excluded as the MitoProt tool revealed the presence of mitochondrial targeting sequence and the cleavage localization

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ACCEPTED MANUSCRIPT signal at the N-terminal region in LdGS protein. (Fig. 5).

402 403

Fig. 5

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3.6. Biochemical characterization of recombinant LdGS enzyme

405

rLdGS enzyme activity was observed within the pH range between 6 and 11, enzyme activity

406

was found to be increasing up to pH 8 after that activity decreases rapidly with increase in pH

407

to 11 (Fig. 6A). Influence of divalent metal ions on the activity of rLdGS protein was checked

408

to see whether there is any specific requirement of a metal ion for its maximal activity. The

409

activity of enzyme increased maximally upon its incubation with Mg2+ followed by Mn2+,

410

Ca2+,Ni2+, and Zn2+ (Fig. 6B). When the purified GS was incubated at different temperatures

411

(5-70 °C) and its activity determined, the GS showed optimal activity at 40 °C. However, the

412

enzyme activity decreases rapidly after 45 °C (Fig. 6C). LdGS activity was detected in lysate

413

of Leishmania promastigotes and it exhibited a specific activity of 322.3µmol/min/mg protein

414

when L-glutamate was used as the substrate while rLdGS show specific activity of 732.5

415

µmol/min/mg protein.

416

The kinetic parameters of the rLdGS were determined by using L-glutamate as the substrate.

417

The reading was taken after 20 min but the enzyme reached saturation in 15 min and no

418

further change in absorbance was monitored. Therefore, the initial velocity of the reaction

419

was monitored over a time period of 20 min as there was linear increase in absorbance.

420

Double reciprocal plots of 1/ LdGS activity versus 1/[ L-glutamate] was linear and revealed a

421

Km value of 26.3 ± 0.4 mM and Vmax of 2.15 ± 0.07 U.mg-1 (Fig. 6D), while double reciprocal

422

plot of ATP revealed Km value of 10.1±1.1 (Fig. 6E).

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Fig. 6

3.7. Effect of GS inhibitors on the growth of L. donovani promastigotes and rLdGS

426

Susceptibility of L. donovani promastigotes towards various inhibitors was assayed. The GS

427

specific inhibitors used in the present study were (L-methionine sulfoximine, L-methionine

428

sulfone, and Phosphinothricin). These are very potent ATP- dependent inactivator of GS from

429

most of the species [50,51]. The concentrations of inhibitors at which 50 % promastigote

430

growth was inhibited (IC50) were found to be more than 100 µM for all compounds (Table.2).

431

Also no significant change in catalytic activity of recombinant LdGS was observed upto 100

432

µM of inhibitor concentration, compared to control (data not shown). Miltefosine was taken

433

as the reference drug, and its IC50 value was 11 ± 1.4 µM which correlates with the

434

previously published results [52]. Therefore in comparison to the standard drug miltefosine

435

these inhibitors are less efficacious.

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436 Table. 2

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4. Discussion

439

Glutamine synthetase catalyzes the synthesis of glutamine from glutamate and ammonia, with

440

hydrolysis of ATP. The product of this reaction, glutamine, takes part in a wide variety of

441

metabolic and synthetic biochemical processes and supports rapidly proliferating cells; for

442

instance, it is a required substrate for ornithine decarboxylase and is an essential precursor for

443

nucleotide biosynthesis. GS is also involved in control of L-asparaginase, glutamate

444

dehydrogenase, proline oxidase, a tryptophan transaminase system and GS itself [53,54]. GS

445

was identified as potential factor for M. tuberculosis pathogenesis and GS in Mtb is involved

446

in the synthesis of the cell wall structure of poly (l)-glutamine found in pathogenic but not in

447

non pathogenic strain of mycobacteria [40]. GS enzyme is important as is evident from the

448

fact that its activity is elevated in brains of patients with Schizophrenia and Alzheimer's

449

disease [55–57]. Glutamine supply is also reported is essential for the growth of some cancer

450

lines [58,59].

451

This is the first report on the molecular characterization of L. donovani glutamine synthetase.

452

This study would provide the basis for further investigation into the molecular function of

453

this gene and exploring this enzyme as potential drug target. The LdGS was successfully

454

cloned and sequenced (GenBank ID: KT907048) and it exhibited maximum identity (78 %)

455

to the putative L. donovani (LDBPK_060370) GS sequence (GenBank ID: XM_003858297).

456

Expression of LdGS in promastigote and axenic amastigote forms of L. donovani parasite was

457

confirmed by western blot analysis. Southern blot studies revealed that it is a single copy

458

gene. Immunolocalization staining showed that LdGS is widely distributed in promastigotes

459

and amastigotes. In both the forms GS is located in cytoplasm. Its presence in mitochondria

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461

predicted by MitoProt tool. It has been reported earlier that in uricotelic vertebrates GS is

462

localized in mitochondria of liver cells and in cytoplasm of brain cells. Also these species

463

contain a single copy of the GS gene [60–62]. In L. infantum GS was predicted to be

464

localized in the mitochondria whereas it is localized in the cytoplasm in L. major due to

465

probable absence of a mitochondrial targeting peptide signal within the sequence [63]. .

466

Influence of divalent metal ions on the activity of rLdGS protein was checked to see whether

467

there is any specific requirement of a metal ion for its maximal activity. The activity of

468

enzyme increased maximum upon its incubation with Mg2+ followed by Mn2+ similar to GS

469

of M. tuberculosis, which also requires magnesium or manganese ions to be in its active state

470

[64]. The activity of rLdGS is relatively stable under pH conditions ranging from 7 to 9,

471

similar to recombinant GS of Mycobacteria and human [64,65]. Also rLdGS shows optimal

472

activity at 40 °C, however the enzyme activity decreases rapidly after 45 °C comparable to

473

recombinant human GS whose midpoint of thermal inactivation was reported to be 49.7 °C

474

[64,65]. The Km value for L-glutamate as a substrate of recombinant glutamine synthetase

475

from Mycobacteria and human has been reported to be 2.56 mM

476

respectively, whereas the Km value of GS from L. donovani is 26.3 ± 0.4 mM in our study

477

[65]. Also Km value for ATP of rLdGS from L. donovani is 10.1 ± 1.1 mM while in case of

478

recombinant human has been reported to be 2.8 mM [65]. The difference in the kinetic

479

parameters between the parasite and human GS indicate that they have different binding

480

affinity with the same substrate L-glutamate indicating that the parasite enzyme can be

481

differentially targeted by designing glutamate or ATP analogs.

482

Inhibitors used in present study are small and highly polar amino acid analogues and the most

483

widely used GS inhibitors are methionine sulfoximine (MSO), methionine sulfone (MS) and

484

phosphinothricin (PPT). MSO and PPT are reported to be phosphorylated in the enzyme

485

active site and subsequently act as transition state analogues [66–68]. MSO has been reported

486

an inhibitor of GS enzyme in various species including mammalian, plant and bacterial GS

487

[69–71]. The concentrations of inhibitors at which 50 % growth of Leishmania promastigote

488

was inhibited were found to be more than 100 µM. These inhibitors did not show any

489

inhibition of recombinant enzyme when tested upto 100 µM concentration suggesting that

490

LdGS specific entities needs to be designed for better efficacy. Treatment of L. mexicana

491

infected macrophages with 5 mM MSO caused effective inhibition of intracellular amastigote

492

proliferation [21]. Also in T. cruzi, glutamate analogs (MSO and MS) showed epimastigote

and 3.5±0.7 mM,

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growth inhibition and the IC50 values estimated at the fifth day of growth were higher for

494

MSO (52.6 ± 4.3 mM) than for MS (17.0 ± 0.3 mM) [17].

495

target the amino acid-binding site, which is highly conserved in both prokaryotic and

496

eukaryotic GS, thus raising selectivity issue [26]. But the nucleotide-binding site is less

497

conserved, thus designing of inhibitors that can bind to this site is more likely to result in

498

selective inhibition [7]. Recently, GS of L. major was modelled and its structure compared

499

with human GS which revealed conservative nature of amino acid binding site however the

500

nucleotide binding site was variant. MtGS inhibitor (MXI) was docked on the ATP binding

501

site yielding good docking scores [72]. Further, in silico analysis and validation of active site

502

residues of LdGS may help in structure based drug designing for treatment of visceral

503

leishmaniasis.

504

This study, include the cloning and expression of LdGS gene in E. coli. Immunolocalization

505

of LdGS was established, and LdGS was characterized biochemically. Copy number of LdGS

506

in promastigotes was confirmed by southern blot analysis. Cytotoxic effect of GS inhibitors

507

on viability of L. donovani promastigotes was studied. Amino acid sequence alignments and

508

the evolutionary tree showed that LdGS shares significant identity with L. mexicana GS, but

509

relatively low identity with the human homologs, suggesting that GS may be a possible new

510

drug target for leishmaniasis.

512

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Amino acid analog inhibitors

Acknowledgements

514

The authors are grateful for the financial support provided by the Ministry of Chemicals and

515

Fertilizers, New Delhi, India. We thank the Director, NIPER, SAS Nagar, Punjab, India for

516

the financial support.

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[39] Y. Si, F. Yuan, H. Chang, X. Liu, H. Li, K. Cai, Z. Xu, Q. Huang, W. Bei, H. Chen, Contribution of glutamine synthetase to the virulence of Streptococcus suis serotype 2, Vet. Microbiol. 139 (2009) 80–88. [40] G. Harth, D.L. Clemens, M.A. Horwitz, Glutamine synthetase of Mycobacterium tuberculosis: extracellular release and characterization of its enzymatic activity., Proc. Natl. Acad. Sci. U. S. A. 91 (1994) 9342–6. [41] H. Chandra, S.F. Basir, M. Gupta, N. Banerjee, Glutamine synthetase encoded by glnA-1 is necessary for cell wall resistance and pathogenicity of Mycobacterium bovis, Microbiology. 156 (2010) 3669–3677. [42] S.H. Fisher, Regulation of nitrogen metabolism in Bacillus subtilis: vive la différence!, Mol. Microbiol. 32 (1999) 223–232. [43] G.J. Crowther, D. Shanmugam, S.J. Carmona, M.A. Doyle, C. Hertz-Fowler, M. Berriman, S. Nwaka, S.A. Ralph, D.S. Roos, W.C. van Voorhis, F. Agüero, Identification of attractive drug targets in neglected- disease pathogens using an in silico approach, PLoS Negl. Trop. Dis. 4 (2010) e804. [44] I. Yeh, R.B. Altman, Drug Targets for Plasmodium falciparum: a post-genomic review/survey., Mini Rev. Med. Chem. 6 (2006) 177–202. [45] A. Debrabant, M.B. Joshi, P.F.P. Pimenta, D.M. Dwyer, Generation of Leishmania donovani axenic amastigotes: Their growth and biological characteristics, Int. J. Parasitol. 34 (2004) 205–217. [46] M.M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem. 72 (1976) 248–254. [47] N. Soumya, I.S. Kumar, S. Shivaprasad, L.N. Gorakh, N. Dinesh, K.K. Swamy, S. Singh, AMP-acetyl CoA synthetase from Leishmania donovani: Identification and functional analysis of “PX4GK” motif, Int. J. Biol. Macromol. 75 (2015) 364–372. [48] J.D. Gawronski, D.R. Benson, Microtiter assay for glutamine synthetase biosynthetic activity using inorganic phosphate detection, Anal. Biochem. 327 (2004) 114–118. [49] T. Mosmann, Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays, J. Immunol. Methods. 65 (1983) 55–63. [50] S.S. Sharma, S.K. Srinivasan, S. Krishnamoorthy, A.M. Kaushal, A.K. Bansal, Preclinical safety pharmacological studies on the amorphous formulation of celecoxib., Arzneimittelforschung. 59 (2009) 254–62. [51] Ł. Berlicki, Inhibitors of glutamine synthetase and their potential application in medicine., Mini Rev. Med. Chem. 8 (2008) 869–78. [52] M.J. Corral, E. González-Sánchez, M. Cuquerella, J.M. Alunda, In vitro synergistic effect of amphotericin B and allicin on Leishmania donovani and L. infantum, Antimicrob. Agents Chemother. 58 (2014) 1596–1602. [53] R.A. Bender, K.A. Janssen, A.D. Resnick, M. Blumenberg, F. Foor, B. Magasanik, Biochemical parameters of glutamine synthetase from Klebsiella aerogenes, J. Bacteriol. 129 (1977) 1001–1009. [54] C. Qiu, Y. Hong, Y. Cao, F. Wang, Z. Fu, Y. Shi, M. Wei, S. Liu, J. Lin, Molecular cloning and characterization of glutamine synthetase, a tegumental protein from Schistosoma japonicum, Parasitol. Res. 111 (2012) 2367–2376. [55] G.S. Burbaeva, I.S. Boksha, M.S. Turishcheva, E.A. Vorobyeva, O.K. Savushkina, E.B. Tereshkina, Glutamine synthetase and glutamate dehydrogenase in the prefrontal cortex of patients with schizophrenia, Prog. Neuro-Psychopharmacology Biol. Psychiatry. 27 (2003) 675–680. [56] E.G. Bruneau, R.E. McCullumsmith, V. Haroutunian, K.L. Davis, J.H. MeadorWoodruff, Increased expression of glutaminase and glutamine synthetase mRNA in

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Figure legends

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Table. 1. Comparison of Leishmania donovani glutamine synthetase amino acid sequence

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with GS sequence from other organisms. 25

ACCEPTED MANUSCRIPT Table. 2. Effect of L-Glutamate analogs, Methionine Sulfoximine (MS), Methionine Sulfone

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(MSO) and Phosphinothricin on L. donovani promastigote.

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Fig. 1. Glutamine synthetase catalyzes the formation of L-glutamine from L-glutamate

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Fig. 2 (A). Multiple sequence alignment of LdGS with GS from other organisms. Sequences

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used in the multiple alignment include: E. coli (CUW82879), M. tuberculosis (NCBI,

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AAB70038), T. brucei (NCBI, XP_846120), T. cruzi (NCBI, XP_820989), L. donovani

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(NCBI, NP_032157.2), L. mexicana (NCBI, AAY45994 ), Z. mays (NCBI, AAM00242), S.

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cerevisiae (NCBI, EDN61170), H. sapiens (NCBI, NP_001028216) and C. familiaris

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(NCBI, AAN41001), (from top to bottom). The number at the beginning of each line

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indicates the residue position in each sequence. Black background indicates sequence

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identity; gray background indicates sequence similarity. The symbol (triangle) indicates

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amino acids (glutamate) binding site, (asterisk) indicates ammonia binding site and (circle)

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indicate ATP binding site. (B) Sequence identity tree using the amino acid sequences of GS

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from L. donovani and other organisms. The tree view program under the CLUSTALW

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program viewed the sequence identity trees derived from the multiple alignments.

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Fig. 3 (A). Diagrammatic representation of the GS locus organization. Black arrows above

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the locus represent the restriction sites within the gene or in flanking areas. The rectangular

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box represents the open reading frame of the GS gene. (B) Southern blot analysis of L.

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donovani glutamine synthetase gene. Lanes 1–6; Restriction digests of L. donovani genomic

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DNA with EcoRI, SalI, NcoI, XhoI, XmaI, and NsiI (internal cutter) respectively. The blot

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was probed with 1140 bp of full-length GS gene.

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Fig. 4 (A). Coomassie stained SDS–PAGE gel of the expressed LdGS protein. M, Broad-

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range molecular-mass marker (Bio-Rad); lane 2-3, uninduced culture pellet and supernatant

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(12 h); lane 4-5, culture pellet and supernatant (0.1mM IPTG, 12 h induction); lane 6-7,

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culture pellet and supernatant (0.5 mM IPTG, 12 h induction); lane 8-9, culture pellet and

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supernatant (1mM IPTG, 12 h induction) (B) SDS-PAGE gel stained with Coomassie

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brilliant blue showing purified samples of LdGS protein by nickel affinity resin ; M, marker,

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lane 2, load; lane 3, flowthrough; lane 4, wash1; lane 5, wash2; lane 6, 7 and 8, purified

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glutamine synthetase of different elution fractions (C) Western blot of recombinant LdGS

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protein using anti-His antibody; M, marker (Bio-Rad); lane 2, supernatant (load); lane 4, flow

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through; lane 5, wash1; lane 6, wash 2; lane 7-9, 10, 8 and 6 µg of purified recombinant

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ACCEPTED MANUSCRIPT 755

LdGS protein respectively (D) Western blot of whole cell extract from amastigote and

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promastigote forms of the parasite using anti-LdGS antibody (left panel). Lane M, molecular-

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mass marker (Bio-Rad); lane 1, amastigote cell extract; lane 2, promastigote cell extract; lane

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3, purified recombinant LdGS protein 5 µg. Ponceau S stained blot in right panel serves as

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loading control.

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promastigotes. Promastigotes were harvested at different time points during growth and

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protein samples were resolved by SDS-PAGE. Western blot shows promastigote cell extract

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at 0 h (lane 1); 24 h (lane 2); 48 h (lane 3); 72 h (lane 4); 96 h (lane 5); and 120 h (lane 6).

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Ponceau S stained blot is shown in lower panel as loading control.

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Fig. 5. GS cellular localization. Immunolocalization of GS in wild type L. donovani

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promastigote (pro) and amastigote (ama) parasites (A and B respectively).

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differential interference contrast image of the parasites, panel (ii) parasites probed with anti-

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LdGS immune sera as primary antibody followed by Alexa Fluor 488 (green) conjugated

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secondary antibody, panel (iii): DAPI (blue) staining of the nucleus and kinetoplast, and

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panel (iv): overlay image of the parasites under the magnification of 100X. Scale bar

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indicates 10µm. The symbols ‘n’ represents nucleus whereas ‘k’ represents kinetoplast.

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Fig. 6. Biochemical characterization of recombinant LdGS protein. (A) Effect of pH, (B)

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metal ion and (C) temperature on LdGS activity. Lineweaver Burk plot showing LdGS

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kinetics with L-Glutamate (D) and ATP (E). Data is represented as mean ± SD of three

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independent experiments.

Panel (i):

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(E) Expression of LdGS along the growth curve of L. donovani

27

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Highlights •

Glutamine synthetase (GS) was cloned from Leishmania donovani and biochemically characterized. GS exists as single copy gene in parasite genome.

•

GS is expressed in both promastigote and amastigote form of the parasite.

•

GS is localized in cytoplasm in both forms of the parasite.

•

GS may be a possible new drug target for Leishmaniasis.

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•