To assess the immunogenicity and antigenic potential of Deubiquitinase from Leishmania donovani and its structural and functional characterization.

Tanishka Kumari

Dept. of Biochemistry, School of Life Sciences, University of Hyderabad, Prof. C. R. Rao Road, Gachibowli, Hydeabad (Telangana) 500046

Prof. Nahid Ali

CSIR - Indian Institute of Chemical Biology, Raja S. C. Mullick Road, Kolkata (West Bengal) 700032

Abstract

Leishmaniasis is a disease caused by obligate intracellular parasite of protozoa group, genus Leishmania. Visceral leishmaniasis (VL) which is also called as Kala- azar or Black fever in Asia is the most severe form of this disease. There is no perfect vaccine or suitable drug to eradicate leishmaniasis completely. So far, no vaccine or drug has been provided to induce long-term protection and ensure effective immunity against leishmaniasis. For clinical diagnosis, invasive methods were being used. Nowadays, for kit-based diagnosis of VL, recombinant kinesin-related antigen like rK39, rK28 and rKE16 are widely used commercially. However, these antigens often show cross-reactivity with endemic healthy controls. Thus, a good diagnostic target antigen is still lacking, so we are trying to identify novel antigens and develop a better diagnostic tool for non-invasive diagnosis of VL. So, our interest is in deubiquitinase (DUB) family as it is a very less studied family in Leishmania. DUBs can cleave the peptide or isopeptide bond between ubiquitin and its substrate protein. Ubiquitination results in degradation of proteins via proteasome, helps in cellular localization of proteins and it also facilitates protein- protein interaction. DUBs play the antagonistic role in this axis by removing these modifications, therefore reversing the fate of the proteins. To check the immunogenicity of DUBs protein, Western blot and ELISA have been performed using one of the antigens from DUBs of Leishmania. It showed positive results with VL patients serum samples and negative with healthy control, endemic healthy control and other disease. This proves that our antigen has high specificity and better diagnostic potential as biomarker of VL. As DUBs family is less characterized in Leishmania, we are interested in its molecular characterization. So, we have performed cloning, expression and purification of DUBs genes. Together this study will help us to identify a novel biomarker for diagnosis and structural and functional characterization of deubiquitinases will pave the way to further validate its therapeutic potential in future.

Keywords: VL, DUBs, ubiquitination, immunogenicity, cloning

Abbreviations

INTRODUCTION

Background

Leishmaniasis is a disease caused by obligate intracellular parasite of protozoa group, genus Leishmania and it is classified as Neglected Tropical Disease (NTD). Transmission of this disease is caused by sand-flies of genus Phlebotomus. This disease is present in three main forms – cutaneous, mucocutaneous and visceral leishmaniasis. Among these three, visceral leishmaniasis (VL) which is also called as Kala- azar or Black fever in Asia, is the most severe form of it and is associated with high fatality. Bangladesh, Brazil, Ethiopia, India, South Sudan and Sudan are the countries where this disease is highly reported and in India most of the cases of VL are found in Bihar.  On the Indian subcontinent, the disease is almost exclusively caused by L. donovani.^[1][4]

Fig 1 Life cycle of Leishmania donovani,   (image source: Leishmania Biology, web.stanford.edu)

Sand-fly carries the infective promastigote stage of parasite when it takes the blood meal from an infected host and inject them into the healthy host during their blood meal. These metacyclic promastigotes convert into aflagellated amastigote after phagocytized by macrophages. Amastigotes multiply in infected cells and affect different tissues.  

Statement of the problem

The diagnosis of VL is complex because its clinical features are shared by a host of other commonly occurring diseases, such as malaria, typhoid, and tuberculosis. Accessibility of the genome sequence of L. donovani has helped to study the expression of many parasite genes and proteins. Based on these studies, recombinant kinesin related antigen, rK39 derived from L. chagasi, has been used widely to make commercial kits for VL diagnosis. But due to its less reliable performance in endemic areas, some better diagnostic markers need to be identified. ^[5]

Objectives of the Research

It should be specific for VL and should not be showing cross reactivity with other diseases and healthy controls even in the endemic regions.

Scope

This study can help us to develop novel antigenic biomarkers for VL diagnosis as well as vaccination. Further characterization and molecular studies of these antigenic candidate and use them as drug target can be helped to design novel and effective drug against VL. Understanding of the mechanisms of host pathogen interaction pathways in detail will help to provide insights into the development of new control strategies against the parasite infection.

LITERATURE REVIEW

Information

Toll Like Receptors (TLR) are family of proteins who plays a role in innate immunity system and work as first line of defence against invading pathogen. It gets activated by surface antigens of pathogen called as ligand and initiates a signalling pathway of inflammatory response for the clearance of pathogen. Binding of ligand and TLRs ultimately recruits IL receptor associated kinase 1 (IRAK1) which will further recruit the TNF receptor associated factor 6 (TRAF6) which triggers its autoubiquitination at Lys-63 residue.

Ubiquitinated TRAF6 activates IKK complex by associating with TGF β associated kinase (TAK 1). NF-κB dimers are activated by IKK-mediated phosphorylation of IκB, which triggers proteasomal IκB degradation. This enables the active NF-κB transcription factor subunits to translocate to the nucleus and induce target gene expression. Product of these genes helps inflammatory response as well as IL 16 and TNFα (proinflammatory cytokines) secretion and combat the effect of infection.^[2]

Although L. donovani has surface glycophospholipid lipophosphoglycan (LPG) which triggers the NF-KB pathway by binding with TLR but it can modulate the TLR pathway and suppress the cytokine production by negatively regulating it.

NF-kB pathway is highly regulated as it results in cytokine overproduction. There are several checkpoints which help in negative regulation of TLR mediated cytokine production. Ubiquitination step of TRAF6 is targeted to regulate the TLR pathway by a deubiquitinase enzyme from pathogen. Host deubiquitinating enzyme A20 normally regulates this step.^[2]

Deubiquitinase (DUBs) is a class of enzyme that cleaves off the bonds between ubiquitin and proteins and regulate ubiquitination mediated pathway. DUBs can be classified into two main classes: cysteine proteases and metalloproteases. The cysteine proteases are divided into 4 superfamilies

1.      Ubiquitin-specific proteases (USPs),

2.      Ubiquitin C-terminal hydrolases (UCHs),

3.      Machado-Josephin domain proteases (MJDs) and

4.      Ovarian tumour proteases (OTU).

The metalloprotease group contains only the Jab1/Mov34/Mpr1 Pad1 N-terminal+ (MPN+) (JAMM) domain proteases.

Deubiquitinase is a well studied protein family in humans but in kinetoplastids it is not well characterized. Among all the deubiquitinase superfamilies, OTU protein family of Leishmania is highly conserved and least similar with human deubiquitinase. In this way this family is our interest of study. The OTU superfamily is a group of cysteine proteases that are homologous to the ovarian tumor (otu) gene product of Drosophila.^[6] This gene codes for a product which is needed for transformation of oogonia into oocyte.^[8] OUT protease contains a catalytic triad of three amino acids Cysteine, Histidine and Aspartate. Anti-inflammatory protein A20 of host showed homology with OTUs. This might be the possible way it is able to modulates the TNF signalling pathway by removing the ubiquitin chain from TRAF6.^[7]

It is reported that OUT like deubiquitinase present in cytoplasm of promastigote form of L.infantum removes lysine 48 (K48)-linked over K63-linked tetra-ubiquitin (Ub) of TRAF6 and thus inhibit NF-kB to get activated. ^[2][3]

Summary

All the informations suggests that deubiquitination step of TRAF6 in TLR mediated NF- κB pathway by Leishmania deubiquitinase enzyme from OUT superfamily is helping the parasite in escaping from host inflammatory response. This might be working as a virulence factor for parasite and helping it to inhibit the inflammatory cytokine release. So, in our study we have worked on the molecular characterization of DUBs from Leishmania donovani to understand their functional role as deubiquitinating enzyme and their importance in disease establishment.

METHODOLOGY

Concepts

For characterization of Dub gene, it was cloned. For cloning Dub gene was amplified by PCR from L.donovani DNA and and transformed in E.coli cells. After successful cloning experiment Dub protein was expressed in E. coli cells by inducing them with IPTG. Expressing Dub protein was further purified by Ni-NTA affinity chromatography. Purified protein was checked for its immunogenicity with VL patients and healthy controls using western blot and ELISA.

Methods

Cloning of Deubiquitinase (Dub) gene in 2 different vector plasmids: pET28a and pET41a

PCR (Polymerase chain reaction)

 Procedure

·         Parasite DNA was isolated from blood by QIAGEN DNA isolation kit.

·         0.8 µL of DNA template was taken in PCR tubes and all the given reagents were added in appropriate volume.

·         PCR tubes were kept in PCR Thermal cycler and program was set as given in the table below

Agarose gel electrophoresis: To visualize the amplified Dub gene

Reagents

·         Agarose powder

·         1X TAE buffer

·         EtBr (10 mg/mL)

·         6X DNA loading dye

Procedure

·         100 mL of 1% agarose gel was prepared in 1X TAE buffer and 8 µL of EtBr was added to it.

·         Gel was casted and comb was used to create wells.

·         10 µL of total sample (2µL dye+ 7µL TAE + 1µL PCR product) was added into the wells. 4 µL of 1kb DNA ladder was added in the first well.

·         Gel was allowed to run for 20 minutes at 120 Volts.

·         After run, gel was visualized in Image lab software of Bio-Rad.

PCR purification

Procedure

·         All the PCR products were pooled in one Eppendorf.

·         5 volume of PB buffer was added and transferred to the column.

·         It was centrifuged at 11,000rpm for 1 minute.

·         Flow through was discarded and column was washed with PE buffer.

·         Absolute Ethanol was added and spanned at 11,000 rpm for 1 minutes.

·         Free spin was given for 2 minutes at the same rpm.

·         50 µL of autoclaved water was added and DNA was eluted in a fresh autoclaved Eppendorf.

Digesion of vector plasmids pET28a AND pET41a and gene of interest (Dub)

·         pET28a, pET41a plasmids and Dub gene both were digested using BamHI and HindIII restriction endonuclease.

·         50 µL reaction mixture was prepared as given below-

                                i.            1 µg of DNA

                               ii.            1 µL of each restriction enzyme

                             iii.           5 µL of CutSmart buffer

                             iv.          ddH₂O to make the volume up to 50 µL

·         Reaction mixture was incubated for 1 hour at 37°C.

·        Enzymes were heat inactivated at 65°C.

·        Digested product was visualized by agarose gel electrophoresis.

Ligation of double digested vectors (pET28a and pET41a) with Dub gene

·         Total 100 ng DNA mixture was prepared for ligation in 3:1 ratio of vector (pET28a and pET41a) and Dub gene insert respectively.

·         Ligation was performed using T4 DNA ligase and buffer at 22°C for 1 hour and kept at 4°C overnight.

Transformation of construct into E. coli cells

Reagents

·         Luria broth media 2.5%

·         LB Agar 2%

·         Kanamycin 50 mg/mL

Procedure

·         1 µL (200-500 ng) of transformed plasmid was added into 50 µL of E.coli cells and kept for 30 minutes in ice.

·         Heat shock was given for 90 seconds at 42°C in dry heat bath.

·         Immediately it was transferred in ice and kept for 10 minutes.

·         1 mL LB media was added to it inside the laminar hood and kept for 1-hour incubation at 37°C.

·         It was centrifuged at 5000 rpm for 5 minutes.

·        900 µL supernatant was discarded and pellet was resuspended in the rest and it was plated on antibiotic kanamycin containing agar plates and kept at 37°C for 16 hours.

·        Bacterial colonies were observed.

Colony PCR

We use colony PCR for rapid screening of bacterial colonies after transformation in pET28a and pET41a vector to verify whether the desired transformed product is present or not. Primers designed to target the insert DNA confirms if the construct carrying our gene of interest (Dub gene).

Procedure

·         A single bacterial colony from both transformed constructs (pET28 a and pET41 a) was picked up with the help of an autoclaved tip and transferred to a PCR tube containing Nuclease free Water. Simultaneously each of those colonies were plated down on kanamycin agar plates.

·         That was used as DNA template and total 20 µL of standard PCR reaction was prepared as mentioned previously.

·         PCR product was run agarose gel and visualize for confirmation.

Plasmid isolation and their digestion with same restriction enzymes to confirm the transformed construct as fall out experiment

Procedure

Those colonies which showed positive results for correct insertion of Dub gene in vector pET28a and pET41a were used for the confirmation of transformed construct.

·         A single colony was picked up and a small 10 mL primary culture was given in LB media containing 0.1% of Kanamycin.

·         Bacterial cells were centrifuged at 5000 rpm for 10 minutes and supernatant was discarded.

·         250 µL of resuspension buffer was added in cell pellet for resuspension of pellet.

·         250 µL cell lysis buffer was added to it and mixed with gentle swirling at room temperature.

·         When the colour of the solution changed to pink completely 350 µL of neutralization buffer was added and kept on ice for 10 minutes.

·         High speed centrifuge was given at 10,000 rpm for 15 minutes.

·         Supernatant was collected and transferred to a fresh column and centrifuged at 11,000 rpm for 1 minute.

·         Flowthrogh was discarded and washed a short spin was given in 500 µL wash buffer at 11,000 rpm for 1 minute. Free spin was given after that.

·         50 µL of Nuclease free water heated at 65°C was added to the column and plasmid was eluted in a fresh autoclaved eppendorf at 11,000 rpm for 2 minutes.

·         This isolated plasmid was double digested with BamHI and HindIII restriction endonuclease.

·         Digested plasmid was checked by agarose gel electrophoresis.

Overexpression of Dub gene at different conditions for standardization of expression protocol

Further we proceeded with Dub gene transformed in Pet28a plasmid. Those bacterial colonies which showed positive results for correct insertion of Dub gene in vector pET28 were used for overexpression of Dub gene.

Reagents

·         Luria broth media 2.5%

·         LB Agar 2%

·         Kanamycin 50 mg/mL

·         1mM IPTG (Isopropyl β-D-1- thiogalactopyranoside)

Procedure

·         A single colony was picked up and a small 10 mL primary culture was given in LB media containing 0.1% of Kanamycin.

·         Primary culture was allowed to grow at 37°C for 16 hours.

·         From primary culture a large 1 Litre secondary culture was given. And incubated in 37°C.

·         When the OD reached 0.6, 0.5M IPTG was given in the culture media for protein expression. Uninduced fraction was also kept.

·         It was incubated for 4 hours at 20°C and 37°C.

·         After that all E. coli. cells that were incubated at different temperatures were pellet down at 8000 rpm for 10 minutes.

·         When the centrifugation was done all different supernatant and pellet fractions were run in SDS-PAGE along with uninduced fraction.

Protein purification

From the different incubation condition standard condition (20°C, 0.6 OD for 2 hours) was selected and Dub protein was overexpressed. Further we proceeded for protein purification.

Reagents

Elution buffer (250 ml)

Elution buffer (250 ml)

It was dissolved in minimum volume; pH was set at 7.5 and 10 % glycerol (25 ml) was added. Volume was made up to 250ml and stored at 4°C

Washing buffer (1 Litre)

Washing buffer (1 Litre)

It was dissolved in minimum volume; pH was set at 8.0 and 10 % glycerol (100 ml) was added. Volume was made up to 1 litre and stored at 4°C.

Resuspension buffer (250 ml)

It was dissolved in minimum volume; pH was set at 8.0 and 10 % glycerol (25 ml) was added. Volume was made up to 250ml and stored at 4°C.

·         PMSF (phenylmethylsulfonyl fluoride) 200mM

·         Lysozyme (30 mg/ml)

Procedure

·         Protein was overexpressed and protein expressing E. coli. cells were pellet down at 8000 rpm for 10 minutes.

·         All the cell pellet was resuspended in total 30 ml resuspension buffer and 1 ml of lysozyme and PMSF was added to it and sonication was performed.

·         After sonication high speed centrifugation was done for 40 minutes at 30,000 rpm.

·         Column was prepared with Ni- NTA beads in 3 ml resuspension buffer.

·         Beads were added in the supernatant and kept at 4°C for 2 hours.

·         Again, column was placed at 4°C room and lysate were allowed to pass through it.

·         Column was washed with washing buffer for 5 times.

·         2 ml of elution buffer was added to the column and protein was eluted in different fractions.

Bradford assay

Reagents

·         Bradford reagent

4 ddH₂O: 1, 5X Bradford reagent

·         Standard solution

2 mg of Bovine serum albumin (BSA) was dissolved in 1 ml of water to make 2 mg/ml standard solution. It was further diluted to make 1 mg/ml, 0.5 mg/ml and 0.25 mg/ml standard solutions

·         Unknown protein

Procedure

·         In an ELISA plate 98 µl of Bradford solution was given in duplicates.

·         2 µl of standard solution and unknown protein solution was added to different wells and mixed with pipette tip.

·         OD was taken at 595nm and standard graph was plotted.

·         Using the equation of the standard graph concentration of our protein was being found.

SDS-PAGE

Materials used in Sample preparation

Sample loading buffer (10 ml)

Sample loading buffer (10 ml)

Destainer

2ddH₂O:2 Glacial acetic acid :1 methanol

Method

·         12% Polyacrylamide gel was prepared using the above listed reagents.

·         25 µl of total sample was loaded in the wells and 4 µl of ladder was loaded in one lane.

·         Gel was allowed to run at 14 mA when the sample is in stacking layer and when it entered the resolving current was increased to 20 mA.

·         When the gel run was done, gel was stained with Coomassie blue stain for 30 minutes and then destained with destainer for 1 hour.

·         Gel was observed in Image lab software of Bio-Rad.

 Parasite lysate preparation

Reagents

Parasite Lysate buffer (pH 7.4)

·         20 mM Tris buffer

·         0.15M NaCl

·         1mM MnCl₂

·         1 mM CaCl₂

·         0.5% Triton x 100

·         0.5% NP 40

Procedure

·         Parasite culture was centrifuged at 3000 rpm for 3 minutes.

·         100 µL of lysis buffer was added in parasite pellet.

·         It was kept on ice for 30 minutes with intermediate mixing with pipette.

·         It was centrifuged at 8000g for 20 minutes, supernatant was collected and kept at -20°C.

Western blot assay

Materials

·         Blocking Buffer (5%)

2.5g of Bovine serum albumin powder was mixed in 50 ml of ddH₂O and filtered through filter paper.

·         10X Tris Buffer Saline

24 g Tris base (formula weight 121.1 g), 88 g NaCl (formula weight 58.4 g) was dissolve in 900 mL distilled water and pH was adjusted to 7.6 with 1N HCl. Distilled was added to make the final volume of 1 Litre.

·         TBST (Tris buffer saline in 0.1 % Tween 20)

500 µl of Tween 20 was added to 500 ml of TBS. 

Procedure

·         12% SDS-PAGE gel was prepared. Protein samples were loaded and allowed to resolve.

Transfer of protein from gel to membrane

·         When the gel run was done resolving layer of gel was taken and placed on the nitrocellulose membrane which is soaked in 1X transfer buffer.

·         It was placed in the blotting cassette between two soaked pads.

·         Set up was closed and extra buffer was removed.

·         Program was set according to mixed molecular weight proteins transfer.

·         Membrane was taken out and stained with ponceau stain for 5 minutes.

·         Membrane was cut and into different strips and transferred to respective vials/ boxes containing 1X TBS.

Blocking of membrane

·         After washing with 1X TBS, 5% blocking buffer was added to the membrane and incubated for 1 hour.

Primary antibody incubation

·         Blocking buffer was removed and washed with 1X TBS 3 times with 5 minutes interval.

·         Patient’s sera were used as a source of primary antibody and given with 1X TBS in 1: 1000 dilution.

·         It was kept for overnight incubation at 4°C.

Secondary antibody incubation

·         After incubation, primary antibody was removed and membrane was washed with TBST 3 times for 10 minutes.

·         Anti-human HRP tagged secondary antibody was given with TBST in 1:1000 dilution and incubated for 2 hours at room temperature.

Blot imaging

·         Secondary antibody was removed and membrane was washed with TBST 2 times and once with 1X TBS.

·         After the washing blot was developed using luminol solution and images were captured in image lab software of Bio-Rad.

ELISA (Enzyme Linked Immunosorbent Assay)

Reagents

·         10X PB (Phosphate buffer)

14.2 g of Na₂HPO₄ was dissolved in 500 ml of distilled water.

7.8 g of NaH₂PO₄ was dissolved in 250 ml of distilled water.

NaH₂PO₄ solution was added to Na₂HPO₄ solution and pH was adjusted to 7.4.

·         10X PBS (Phosphate buffer saline)

14.2 g of Na₂HPO₄ was dissolved in 500 ml of distilled water.

7.8 g of NaH₂PO₄ was dissolved in 250 ml of distilled water.

NaH₂PO₄ solution was added to Na₂HPO₄ solution and pH was adjusted to 7.4.

9% NaCl was added to this solution.

·         PBST (PBS in 0.05% Tween 20)

500 µl of Tween 20 was added to 1000 ml of 1X PBS.

·         1% Blocking Buffer

10 ml of 5% BSA was added to 40 ml of 1X PBS to make total 50 ml of 1% Blocking buffer.

·         Substrate solution

O- Phenylenediamie dihydrochloride (OPD) with H₂O₂ (Hydrogen peroxide) or

TMB (3,3′,5,5′-Tetramethylbenzidine) solution

·         Stopper solution

2 N H₂SO₄

Procedure

·         1 µg of protein was coated per well in 100 µl of 1X PB buffer and kept for overnight at 4°C.

·         Phosphate buffer was removed and wells were washed with 1X PBST FOR 3 times.

·         After washing 200 µl of 1% BSA was used for blocking of each well for 2 hours at 37°C.

·         After this incubation wells were washed with 1X PBST for 3 times.

·         100 µl of primary antibody solution (VL Patient’s and healthy patient’s sera in 1: 2000 dilution in 1X PBS) was added in 94 wells in duplicates.

·         100 µl of 1X PBS was given as blank in last well in duplicate.

·         Plate was kept for incubation at 37°C for 2 hours.

·         After this plate was washed for 3 times with 1X PBST.

·         Anti-human HRP tagged secondary antibody was given in 1: 3000 dilution (100 µl each well) and kept for 1-hour incubation at 37°C.

·         After 3 washes with 1X PBST 100 µl of substrate solution was given in all 96 wells.

·         After 5-10 minutes reaction was stopped using 50 µl per well stopper solution.

·         OD was taken at 450 nm in ELISA plate reader program.

RESULTS AND DISCUSSION

Cloning of Dub Gene

Fig 2 PCR amplified Dub gene (702bp) from genomic DNA of L.donovani at different annealing temperature

where, M – DNA ladder

L1 – Dub gene amplfied at 65°C anealing temperature

L2 – Dub gene amplfied at 64.3°C anealing temperature

L3 – Dub gene amplfied at 63°C anealing temperature

L4 – Dub gene amplfied at 61.1°C anealing temperature

L5 – Dub gene amplfied at 58.8°C anealing temperature

L6 – Dub gene amplfied at 55.7°C anealing temperature

Fig 3 Transformed Dub gene (702bp) in pET28a plasmid amplified by Colony PCR

Where M – DNA ladder and L1 to L14 are showing different E. coli colonies

Fig 4 . Transformed Dub gene (702 bp) in pET41a plasmid amplified by Colony PCR

Where M – DNA ladder and L1 to L16 are showing different E. coli colonies

Fig 5 Fall out product of transformed Dub gene(702bp) in pET28a(5.4bp) plasmid after double digestion with BamHI and HindIII

Where M- DNA ladder and L1 to L7 are showing fall out products

Fig 6 . Fall out product transformed Dub gene(702bp) in pET41a (5.9k bp) plasmid after double digestion with BamHI and HindIII

Where M- DNA ladder and L1, L3, and L5 are showing fall out products

Fig 7 7 Overexpressed Dub protein at different conditions

L1 – supernatant of uninduced fraction

L2 – supernatant of bacterial culture from pET28a transformed Dub gene grown at 20°C for overexpression

L3 - pellet of bacterial culture from pET28a transformed Dub gene grown at 20°C for overexpression

L4 - supernatant of bacterial culture from pET41a transformed Dub gene grown at 20°C for overexpression

L5 – pellet of bacterial culture from pET41a transformed Dub gene grown at 20°C for overexpression

L6 - supernatant of bacterial culture from pET28a transformed Dub gene grown at 37°C for overexpression

L7 – pellet of bacterial culture from pET28a transformed Dub gene grown at 37°C for overexpression

L8 – supernatant of bacterial culture from pET41a transformed Dub gene grown at 37°C for overexpression

L9 - pellet of bacterial culture from pET41a transformed Dub gene grown at 37°C for overexpression

Fig 8 Purified Dub protein from Ni- NTA affinity chromatography

Where, M- Protein marker and L1 to L5 are different elution of purified protein 

Immunoblot Assay

Fig 9 Dub protein probed against unimmunized mice sera (L1) and immunized mice sera (L2)

Fig 10 Dub antibody probed against L. donovani lysate (L1)

Fig 11 9 VL infected sera samples (L1 TO L9)

Fig 12 9 Patient’s sera samples including VL infected sera (L1), 2 healthy controls (L2 and L3), 2 Endemic healthy control (L4 and L5) and 4 other diseases (L6 Malaria, L7 Viral fever, L8 Typhoid and L9 Tuberculosis).

ELISA

Fig 13 ELISA results from sera of VL pateints and other controls. Comparitive evaluvation of anti- Leishmania IgG antibodies in serum samples against Dub gene at 0.39 cut-off. In the study panel we had VL patients (n= 38), Healthy controls (n=4), Endemic healthy controls (n=5) and Other diseases (n=6).

Fig 14 Receiver operator characteristics (ROC) curve for serum ELISA. ROC curve obtained from the ELISA values for detection of anti- Leishmania IgG antibodies in serum samples against Dub gene cut-off value (0.3985), senstivity (97.37%), specificity (100%) were obtained by this curve using GraphPad Prism software.

Fig 15 ELISA results from sera of VL pateints and other controls. Comparitive evaluvation of anti- Leishmania IgG1 antibodies in serum samples against Dub gene at 0.1485 cut-off. In the study panel we had VL patients (n= 13), Endemic healthy controls (n=4) and Other diseases (n=3).

Fig 16 Receiver operator characteristics (ROC) curve for serum ELISA. ROC curve obtained from the ELISA values for detection of anti- Leishmania IgG1 antibodies in serum samples against Dub gene cut-off value (0.1485), senstivity (76.92%), specificity (100%) were obtained by this curve using GraphPad Prism software.

Fig 17 ELISA results from sera of VL pateints and other controls. Comparitive evaluvation of anti- Leishmania IgG2 antibodies in serum samples against Dub gene at 0.1318 cut-off. In the study panel we had VL patients (n= 13), Endemic healthy controls (n=4) and Other diseases (n=3).

Fig 18 Receiver operator characteristics (ROC) curve for serum ELISA. ROC curve obtained from the ELISA values for detection of anti- Leishmania IgG2 antibodies in serum samples against Dub gene cut-off value (0.1318), senstivity (38.46%), specificity (100%) were obtained by this curve using GraphPad Prism software.

Cloning experiment was performed using Dub gene. Firstly from Leishmania DNA Dub gene was polymerized using Dub specific primers at different annealing temperatures. Dub gene was successfully polymerized in all those temperatures used to anneal primers (Fig.2). This amplified gene was transformed in two different vectors pET28a and pET41a and cultured bacterial colonies were analysed for the confirmation transformed construct by Colony PCR(Fig.3 and Fig.4). Positive result showing colonies were further analysed by fall out experiment and showed two bands representing plasmid and gene and confirmed correct gene insertion in right orientation (Fig.5 and Fig.6).

After successful cloning of Dub gene it was overexpressed in E.coli cells at different incubation conditions for standardization of overexpression protocol for our gene in both the vectors (Fig.7). Overexpression was standardize for Dub gene at 0.5mM IPTG induction, at 20°C for 4 hours as this condition favors the better expression for our protein.

Overexpressing protein in pET28a vector was purified with Ni-NTA affinity chromatography as it contains 6 residues of His-tag which can bind to Ni-NTA beads and further eluted in high (250mM) immidazole concentration (Fig.8).

Anti-Dub antibodies were generated in mice and tested against Dub antigen and parasite lysate by immunoblot assay. Bands were found showing positive results for antibody interaction with purified gene as well as with parasite lysate confirming the presence of Dub antigen in the lysate (Fig. 9 and Fig 10).

In immunoblot assay 9 VL patient’s sera was used against our purified Dub protein and tested for the presence of antibody in the sera against our protein. Dub protein showed reactivity with all 9 VL infected sera (Fig 11). Reactivity of Dub protein was also tested with healthy control, endemic healthy control and other diseases including malaria, viral fever, typhoid and tuberculosis. Antibodies against Dub protein were completely absent in our protein and showed no cross-reactivity (Fig 12).

In ELISA also same type of study was done with large number of VL positive sera samples and other controls. VL infected serum samples showed very high OD value compare to healthy controls and endemic healthy controls. This data showed high sensivity (97.37%) of IgG Dub gene against Dub gene and it showed high specificity (100%) with VL samples in comparision with other control samples.(Fig 13 and Fig 14).

Along with this, ELISA was performed to see the level of anti-Leishmania antibodies from IgG sub-classes. In this data we found that IgG1 showed better senstivity (76.92%) against Dub protein (Fig.15 and Fig 16) whereas IgG2 showed very less senstivity (38.46%).ELISA results for IgG2 showed almost equal values for VL and other controls (Fig17 amd Fig18).

CONCLUSION

In our study we performed immunoblot assay and ELISA to study the immunogenicity of Dub protein against VL patients sera and also tested with healthy controls and other disease and we found that our protein has been worked as an effective diagnostic biomarker and showed high senstivity and specificity. Detailed structural analysis of different domains of this protein will further help us to understand its active site and its effect on parasite viability when mutated or knocked out. Through this way we can use this protein as a therapeutic drug target to combat the effect of infection.

REFERENCES

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ACKNOWLEDGEMENTS

To speak gratitude is courteous and pleasant, to enact gratitude is generous and noble, but to live gratitude is to touch Heaven. I would like to admit that it would have been difficult if not impossible for me to complete this study without support and encouragement of the people to whom I am about to express my gratitude.

I would express my gratitude to Indian Academy Sciences for having given the opportunity to me to work as Summer Research Fellow.

I would like to thank the Director, Prof. Samit Chattopadhyay, of Indian Institute of Chemical Biology for allowing me to do my summer project in this esteem institution.

I wish to place on record, my sincere gratitude to Prof. Nahid Ali for accepting me to work under her guidance. I would like to thank her for providing me with a lot motivation throughout the project.

I am extremely grateful to Mr. Mohd. Kamran for their invaluable guidance and great patience during the period of my training.

It also gives me immense pleasure in expressing my gratitude to my lab members. I would like to thank Ms. Sonali Das for her constant help and support, I would express my regards to Ms. Nicky Didwania for sharing her invaluable work experience to guide me in all the experiments and I would like to thanks Mr. Anirban Bhattacharya for his help and to always make the lab environment cheerful. I would also like to thank my summer fellow mates Sohitri Mukherjee and Jagriti Das for giving me wonderful company throughout the stay, without you both this internship would have never been memorable for me.

I would like to extend my genuine gratitude to, Ms. Sneha Ghosh, Dr. Sarfaraz Ahmad Ejazi, Dr. Abdus Sabur, Dr. Somsubhra Thakur, Dr. Mithun Maji and Mr. Sumon Midya for their help and support.

Finally, I also express my deep thanks to my parents for their support and encouragement.

To speak gratitude is courteous and pleasant, to enact gratitude is generous and noble, but to live gratitude is to touch Heaven. I would like to admit that it would have been difficult if not impossible for me to complete this study without support and encouragement of the people to whom I am about to express my gratitude.