study of the enzymatic and immunological properties of glycylpeptide N-tetradecanoyltransferase isolated from Leishmania By: 1. Saif Qahtan Salman (Lecturer of Biochemistry, College of Biotechnology, Al-Qasim Green University, Babylon, Iraq, E-mail: saif.q1988@gmail.com) 2. Karam Dawood Salman (Lecturer of Immumopathology, College of veterinary medicine, Al-Kufa University, Kufa, Iraq, E-mail: karam.biotech@mail.com) 3. Ghufran A. Abdulraheem (Lecturer of Zoology, College of Biotechnology, Al-Qasim Green University, Babylon, Iraq, E-mail: gogo868776@yahoo.com) Al-Qasim Green University Iraq, 2015 Abstract Visceral leishmaniasis caused by Leishmania donovani is the most severe systemic form of the disease. There are still no vaccines available for humans and there are limitations associated with the current therapeutic regimens for leishmaniasis. Recently, we reported functional importance of glycylpeptide N-tetradecanoyltransferase enzyme from L. donovani involved in ascorbate biosynthesis pathway. In this study, we have shown that glycylpeptide Ntetradecanoyltransferase have many immunological properties can act as enhancer for host’s immune system and can work like leishmanial antigen after studying enzymatic enzymatic and immunological properties of this enzyme on 4 to 6 weeks old BALB/c mice . Cytosolic antigen was prepared by repeated freezing- thawing of leishmanial antigen, followed by ultrasonication and ultracentrifugation. The protein concentration was estimated by Lowry method and cytosolic protein was characterized over SDS-PAGE gel with dominant polypeptides ranging from 96 to 27 kDa . Cytosolic antigen was administered subcutaneously along with chitosan at 2 weeks interval. 10 days postimmunization co-relates for cell mediated- and humoral- immune response were evaluated. Successful vaccination for the control of parasite multiplication is often related to antigen induced DTH response as an indication of activation of cell-mediated response. In the present study, results obtained upon immunization with cytosolic antigen in association with chitosan demonstrated induction of an appreciable DTH response suggesting the activation of cell-mediated immunity. Activation of cell mediated immunity was further evaluated in terms of lymphocyte proliferation. Lymphocytes from both spleen and lymph node were in vitro restimulated with cytosolic antigen. Lymphocytes exhibited a strong in vitro recall response where proliferation induced in response to cytosolic protein and cytosolic protein in combination with chitosan was almost comparable . Further, induction of nitric oxide (NO) production in splenic lymphocytes was studied as production of NO molecules is an important cytotoxic function that macrophages used to resolve infection by several obligate intracellular protozoa and bacterial parasites. Immunization of cytosolic protein in association with chitosan resulted in considerable increase in NO production in comparison unimmunized, cytosolic and chitosan alone immunized groups respectively . Through these results we saw glycylpeptide Ntetradecanoyltransferase can induce protective immunity and can provide sustained protection against Leishmania donovani. We further conclude that the parasites attenuated in their antioxidative defence mechanism can be exploited as vaccine candidates Key words: Leishmania donovani, glycylpeptide N-tetradecanoyltransferase, SDS-PAGE. Introduction Leishmania are protozoan parasites shuttling between sand fly vector where they multiply as free promastigotes in the gut lumen, and mammalian host where they proliferate as obligatory intracellular amastigotes in the mononuclear phagocytes ( Handmanet al., 1999 ).Leishmania parasites are responsible for a family of diseases, collectively known as leishmaniasis, with discrete clinical features ranging from cutaneous lesions to a fatal systemic disease. Leishmaniasis is prevalent in Africa, Latin America, Asia, the Mediterranean basin and the Middle East, and recently has been identified in East Timor(Chevalier et al., 2000 ) , Thailand kangaroos in Australia (Rose et al., 2004) . (Kongkaewet al.,2007) and in Leishmaniasis has been classified as one of the most neglected diseases, and the estimated disease burden places it second in mortality and fourth in morbidity among the tropical infections. For many years, the public health impact of leishmaniasis has been underestimated, as a substantial number of cases were never recorded. The expansion of leishmaniasis and the sharp rise in prevalence is related to environmental changes and migration of non-immune people to endemic areas (WHO Initiative for Vaccine Research 2008). The former, in particular, has the potential to expand the geographic span of the vector, thus increasing Leishmania transmission to previously unaffected areas. More recently, an increase in the overlapping of HIV infection and visceral leishmaniasis has been observed, especially in intravenous drug users in South-Western Europe and Brazil(Cruz et al., 2006). The situation might be much worse in Africa and Asia where the prevalence and detection of HIV and Leishmania coinfections is still largely underestimated. Visceral leishmaniasis (VL), also known as kala-azar, is a disseminated protozoan infection caused by Leishmania donovani complex (Chappuiset al.,2007;Murrayet al.,2005). VL is essentially caused by L.donovani and Leishmania infantum (synonym Leishmania chagasiin South-America). Exceptionally, visceralization of species typically associated with cutaneous leishmaniasis has been observed. Most commonly, this has been reported with Leishmania tropical in the Middle East and Leishmania amazonensis in South-America. In individuals infected with human immunodeficiency virus (HIV),visceralization of a number of dermatotropic species has been documented as well (see section on VL-HIV coinfection. Zoonotic visceral leishmaniasis (ZVL) caused by Leishmania infantum is a disease of humans and domestic dogs (the reservoir) transmitted by Phlebotomine sandflies. According to the World Health Organization (Chaniotiset al., 1994), ZVL was first recorded on Crete in 1907, after which it featured prominently in medical literature as a serious public health problem. In Chania, Crete, the annual incidence in the 1930s was 50 cases/30,000 population(Adler et al., 1938), and 21% of 1,115 dogs were positive for ZVL by the formol-gel serologic test; 30% of those were symptomatic (Chaniotiset al., 1994). After World War II, use of DDT against malaria vectors and focal destruction of Leishmania spp.–infected dogs is thought to have reduced ZVL on Crete. Sandflies were not found in villages systematically sprayed during 1946–1949 compared with unsprayed villages (Hadjinicolaou 1958). During 1951–1975, only 33 alleged human ZVL cases were recorded on Crete (Leger et al., 1977), and reports of canine ZVL were scanty. In 1983 a sero survey of 72 stray dogs in Chaniaidentified only 1 infected dog (Chaniotiset al., 1994). By the late 1980s, Phlebotomus neglectus, the putative vector of L. infantum on Crete, was abundant in stonewalls inside and outside villages around Heraklion(Leger et al., 1993).During 1999–2004, P. neglectuswas found in abundance in human dwellings and rural locations (hollows in olive trees and near rodent burrows). Since 1991, 38 persons who came to hospitals in Heraklion were confirmed as having cases of ZVL. Today, canine infection is confirmed throughout the island (M. Antoniou, unpub. data), and seroprevalences(30%–40%) are some of the highest reported in Europe. These accounts anecdotally suggest that the incidence of ZVL has increased on Crete during the past decade, which, if so, would be relevant to the public and veterinary health sectors. Meterials and methods - MICE AND PARASITES BALB/c mice, bred in the animal care facility of the University under pathogen-free conditions, were used at 4 to 6 weeks old for experimental purposes with prior approval from the Animal Ethics Committee of the JamiaHamdard. An Indian strain of L. donovani AG83 was maintained by passage in BALB/c mice, amastigotes isolated from infected mice spleens were allowed to transform to promastigotes by cultivation at 22°C in M199 medium (pH 7.4) supplemented with 20% heat-inactivated FBS, 2 mM glutamine, 25 mM HEPES, 100 U/ml of penicillin G-sodium and 100 µg/ml of streptomycin sulfate. Parasites were checked and enumerated by counting in a hemocytometer on day 6 after first transformation and every 72 hrs thereafter. Designated parasites from stationary-phase cultures were diluted in fresh medium with the same composition as that mentioned above to maintain an average density of 2 x106 cells/ml (Afrin et al, 2002). Determination of antigen concentration by Lowry’s method Principle The “Lowry Assay: Protein by Folin’sReagent” (Lowry et al., 1951) has been the most widely used method to estimate the amount of protein (already in solution or easily-soluble in dilute alkali) in biological samples. First the proteins are pretreated with copper ion in alkali solution, and then the aromatic amino acids in the treated sample reduce the phosphomolybdatephosphotungstic acid present in the Folin’s Reagent. The end product of this reaction has a blue color. The amount of protein in the sample can be estimated via reading the absorbance (at 750 nm) of the end product of the Folin reaction against a standard curve of a selected protein solution ( in our case; Bovine Serum Albumin(BSA) solution). The Lowry method relies on two different reactions: 1. The first reaction is the formation of a copper ion complex with amide bonds, forming reduced copper in alkaline solutions. This is called a Biuret chromophore and is commonly stabilized by the addition of tartarate(Gornallet al., 1949). 2. The second reaction is reduction of the Folin-Ciocalteau reagent (phosphomolybdate and phosphotungstate), primarily by the reduced copperamide bond complex as well as by tyrosine and tryptophan residues. PREPARING THE SOLUTION The Lowry Solution is a mixture of the ( Na2 Tartrant.2(H2O), CuSO4.5(H2O) and Other NaOH, Na2CO3) chemicals, except the Folin’s Reagent. The Lowry solution should be prepared fresh, at the day of measurement. Though the individual solutions for the Lowry solution can be prepared in advance and then mixed at the day of measurement. Solution A is a dilute alkali solution. 2N Folin and Ciocalteau’s Phenol Reagent contain HCl and H2PO4. Solution A: (alkaline Solution) (for 100 ml) 0.572gm NaOH 2.862gm Na2CO3 Solution B: (for 20 ml) 0.285gm CuSO4.5(H2O) Solution C: (for 20 ml) 0.571gm Na2Tartrant.2(H2O) Lowry Solution: (freshly prepared, 0.7/ml sample) Sol. A + Sol. B +Sol. C with a ratio (vol:vol) of 100:1:1 Folin Reagent: (instant fresh, 0.1 ml/sample) 5ml of 2N Folin and Ciocalteu’s Phenol Reagent + 5ml of double distilled water this solution is light sensitive. So it should be prepared at least 5min of the first sample incubation and kept in an amber container. Procedure: 1. Different dilutions of BSA are prepared by mixing stock BSA (1 mg/ml) and water in the test tube. Similarly, two dilutions of antigen are prepared in duplicate .The final volume in each of the test tubes is 5 ml. The BSA concentration ranges from 0.05 to 1 mg/ml. 2. From these different dilutions, pipette out 0.2 ml BSA or antigen to different test tubes and add 2 ml of alkaline copper sulphate reagent. Mix the solutions well. 3. This solution is incubated at room temperature for 10 mins. 4. Then add 0.2 ml of reagent Folin Ciocalteau to each tube and incubate for 30 min. Zero the colorimeter with blank and take the optical density (measure the absorbance) at 660 nm. 5. Plot the absorbance against BSA concentration to get a standard calibration curve. 6. Check the absorbance of antigen and determine the concentration of the unknown sample using the standard curve. SDS-PAGE Principle Electrophoresis is the study of the movement of charged molecules in an electric field. The generally used support medium is cellulose or thin gels made up of either polyacrylamide or agarose. Cellulose is used as support medium for low molecular weight biochemicals such as amino acid and carbohydrates whereas agarose and polyacrylamide gels are widely used for larger molecules like proteins. The general electrophoresis techniques cannot be used to measure the molecular weight of the biological molecules because the mobility of a substance in the gel is influenced by both charge and size. In order to overcome this, if the biological samples are treated so that they have a uniform charge, electrophoretic mobility then depends primarily on size. The molecular weight of protein maybe estimated if they are subjected to electrophoresis in the presence of a detergent sodium dodecyl sulfate (SDS) and a reducing agent mercaptoethanol ( ME). SDS disrupts the secondary, tertiary and quaternary structure of the protein to produce a linear polypeptide chain coated with negatively charged SDS molecules. 1.4grams of SDS binds per gram of protein. Mercaptoethanol assists the protein denaturation by reducing all disulfide bonds. SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) Polyacrylamide gels are prepared by the free radical polymerization of acrylamide and the cross linking agent N N’ methylene bis acrylamide. Procedure: Casting the gels 1. Prepare the separating gel monomer solution by combining all reagents except ammonium persulfate (APS) and TEMED. Deaerate and mix the solution after adding each reagent by swirling the container gently. 2. Place a comb completely into the assembled gel sandwich. With a marker pen, place a mark on the glass plate 1 cm below the teeth of the comb. This will be the level to which the separating gel is poured. Remove the comb. 3. Add APS and TEMED to the monomer solution and mix well by swirling gently. Pipette the solution to the mark. 4. Immediately overlay the monomer solution with 1 ml. of water. Use a steady, even rate of delivery to prevent mixing with the gel. 5. Allow the gel to polymerize for 45 minutes to 1 hour. Pour the water overlaying the gel and drain the excess water with strips of filter paper. 6. Prepare the stacking gel monomer solution. Combine all reagents except APS and TEMED. Deaerate and mix the solution by swirling gently. 7. Place a comb in the gel sandwich. 8. Add APS and TEMED to the solution and pipette the solution down one of the spacer until the sandwich is filled completely. 9. Allow the gel to polymerize for 15 minutes. 10. Remove the comb. 11. Gel is placed in the buffer chamber and running gel buffer is added into the chamber . Preparation of a 10% Resolving/separating gel. 30% acrylamide + 0.8% Bisacrylamide 1.25 ml 4X Tris-HCl/SDS, pH 8.8 0.94 ml Distilled water 1.56 ml 10% ammonium persulfate (APS) 25 l TEMED 10 l Preparation of a 4.5% stacking gel 30% Acrylamide + 0.8% Bis-acrylamide 0.65 ml 4X Tris-HCl/SDS, pH 6.8 1.25 ml Distilled water 3.05 ml Ammonium persulfate 25l TEMED 5 l The Leishmanial antigen (Cytosolic) from L. donovani promastigotes was subjected to sodium dodecyl sulphate 10% polyacrylamide gel electrophoresis (SDS–10% PAGE), and the proteinswere localized in gels, stained with Coomassie blue . (Ulrich K. Laemmli , 1970 ) Evaluation of Cell Mediated Immunity DTH (delayed type hypersensitivity): The delayed type hypersensitivity was the first experimental evidence of transferable immunity carried only by the immune cells. Robert Koch, the discoverer of the tubercule bacillus was the first to demonstrate a delayed type hypersensitivity reaction in 1882. Koch attempted to use his killed tuberculin preparation as a prophylactic and therapeutic vaccine. Unfortunately, the antigen did not confer protection to naive patients, and when injected intravenously in infected patients, caused reactivation of the disease and in some cases death. Nevertheless, when the antigen was injected intradermally, the delayed inflammatory response (tuberculin reaction) could indicate whether or not an asymptomatic person had been exposed to Mycobacterium tuberculosis. It was not until 1942 that Landsteiner and Chase demonstrated that the DTH reaction could be transferred by a "cell only" fraction. The basis for the experiment was fairly direct. Cells from guinea pigs, which had been immunized with Mycobacterium tuberculosis or hapten, were transferred into naive guinea pigs. Later, when antigen or hapten was injected into these guinea pigs, they elicited an immune recall response that was not present in the naive controls. This did not happen when the serum fraction was transferred. Coombs and Gell classified delayed type hypersensitivity as Type IV. The three other classifications of hypersinsitivity are Type I (immediate/IgE-related) in which the cutaneous skin test reaction peaks at 2 hours, Type II (antibody and complement related cytotoxicity) and type III (antigenantibody complex mediated). DTH responses have been well characterized. The reaction is antigen specific and causes erythema and induration at the site of antigen injection in immunized animals or humans. Systemic injection of antigen results in fever, synthesis of acute phase proteins and in some instances death. The nature of this antigen can be varied. Mycobacteria, protein, hapten, and even grafted tissue are all capable of inducing delayed type hypersensitivity reactions. The histology of DTH can be different for different species, but the general characteristics are an influx of immune cells at the site of injection, either macrophages and basophils in humans and mice or neutrophils in guinea pigs, and induration which becomes apparent within 24-72 hours. Even though they make up only a small percentage (10-20%) of the total inflammatory infiltrate at 48 hours, T cells (either CD4+ or CD8+ depending on the antigen) are required to initiate the reaction. Evaluation of DTH Response: The response was evaluated at 24 h by measuring the difference in swelling between two hind footpads, one injected with 50 µl of PBS alone and the other with PBS containing L. donovani antigens (cytosolic antigen) (40 µg/50 µl PBS), thickness of foot pad swelling measured by using a digital Vernier caliper .Lymphocyte proliferation by Trypan blue exclusion method BALB/c Mice sacrificed Isolation . Spleen Lymph Nodes Making single cell suspension by frosted (sterile) Making single cell suspension by pestle and mortar (sterile) RBCs lysed by lysis buffer Wash 2 times with RPMI 1640 without PR and FBS Wash 2 times with RPMI 1640 without PR and FBS Adjust cell density to 5x106 cells/ml with complete RPMI 1640 after trypan blue exclusion and counting under phase contrast microscope Adjust cell density to 2x106 cells/ml with complete RPMI 1640 after trypan blue exclusion and counting under phase contrast microscope Cultured in RPMI 1640 containing 20 mM NaHCO3, 10 mM HEPES, 100U of penicillin per ml, 100 µg of streptomycin per ml, 2 mM L-glutamine, and 10% fetal calf serum (complete medium [CM]), with 50 μMβmercaptoethanol. added. The cells were cultured in triplicate in a final volume of 200 μl/well with cytosolic antigen (12μg/ml) at optimum concentrations, Normal cells along with Antigen ,or with ConA (mitogen), cells from chitosan alone , and Antigen along with chitosan immunized mice. The cultures were incubated for 48 h at 37°C in a humidified chamber containing 5% CO2. The amount of proliferation was measured by counting the cells after Trypan blue exclusion using hemocytometer (Poulteret al.,1982) Nitric oxide estimation Production of NO molecules is an important cytotoxic function that macrophages use to resolve infection by several obligate intracellular protozoa and bacterial parasites. The inducible form of the NO Synthase enzyme (iNOS) produces large amounts of reactive nitrogen molecules by oxidizing the terminal guanidino nitrogen of L- arginine. Since reactive nitrogen molecules can damage host tissues as well as the invading microbes, the activation of NO production by macrophages is a tightly regulated ligand receptor mediated process that involves two stages. The priming stage occur when activating molecules, such as the cytokine INF-ϒ, binds to receptors on the cell surface of macrophages, inducing alterations in cytoplasmic calcium levels and initiating other intracellular signaling cascades. These signaling cascades ultimately alter the expression of enzymes, including iNOS and receptor proteins to prepare the metabolic machinery to initiate a cytotoxic response. The triggering stage is induced when another molecule often a molecule of the intruding microbe such as bacterial LPS binds to the receptor on macrophages initiating another series of signaling cascade that activates the previously primed cytotoxic machinery. Nitric Oxide Assay involves the conversion of nitrate to nitrite by the enzyme nitrate reductase. The detection of nitrite is then determined as a coloured azo-dye product of the Griess reaction that absorbs visible light at 540 nm. The concentration of NO can be extrapolated from a standard curve of sodium nitrite. The Griess reaction involves an oxidation and a nucleophilic reaction. Buffer or sample components that interfere with the oxidation and nucleophilic reaction may interfere with color formation. Examples of nucleophiles and antioxidants are azide, ascorbic acid, compounds containing sulphydryl groups such as cysteine, glutathione, DTT, and β-mercaptoethanol. Nitrate Reductase converts nitrate to nitrite. Any sample or buffer component that may interfere with this enzyme will lower the conversion of sample nitrate to nitrite and therefore result in lower estimates of NO. The nitrate salt content should be considered when choosing a tissue culture media. Some media may contain relatively high levels of nitrate, which may interfere with sensitive detections. Media that contain phenol red as a pH indicator do not interfere with the Griess reaction as the indicator is typically yellow colored under the conditions of the Griess reaction. Preparation of solution: Reagent A:- for 10ml 1 sulphanilamide (0.1gm) in 1.2 N HCl (1.08 ml). Reagent B:- for 10ml 0.3 NEDD (1-naphthyl ethylene diaminedihydrochloride) (30mg) in milli Q water. Reagent C:-for 10ml 0.1M NaNO2 (6.9mg) in milli Q. Griess reagent:1 sulfanilamide and 0.1 NEDD in 50 H3PO4 Determination of nitric oxide The spleens were removed aseptically and teased into single cell suspension in RPMI 1640 supplemented with 10% FBS, 2mM L-glutamine, Penicillin (100 U/ml), Streptomycin (100µg/ml) and 50µM 2-mercaptoethanol. RBCs were removed by lysis with 0.14M Tris buffered NH4Cl. The remaining cells were washed twice with culture medium and viable mononuclear cell number was determined by counting after Trypan blue exclusion method in a haemocytometer. The cells from the immunized and normal mice were stimulated with soluble leishmanial antigen at a concentration of 5g/ml, cultured in complete medium at 2x105 cells/well in a 96 well plate flat bottom culture plate. ConA was taken as positive control at a concentration 2.5µg/ml. after incubation for 48 hrs in CO2 incubator nitrite concentrations were determined in the culture supernatants using Greiss reagent. Equal volumes (100 L) of cell culture medium and Greiss reagent were mixed and incubated at room temperature, and absorbance was measured 550 nm with a microplate reader. Nitrite concentration was determined using a standard curve generated with sodium nitrite. Results Determination of antigen (cytosolic) concentrations by Lowry’s method The concentration of cytosolic antigen was determined by Lowery’s method using BSA as a control to generate standard curve (Fig.14) and the concentration of cytosolic antigen was found to be 3.32 mg/ml. 1.4 y = 0.012x R² = 0.9693 1.2 OD (750 nm) 1 0.8 0.6 0.4 0.2 0 0 20 40 60 80 100 120 Concentration (µg/ml) Fig.14 BSA standard graph for estimation of protein concentration CHARACTERIZATION OF ANTIGEN (SDS-PAGE) The protein profile was examined by SDS-PAGE. SDS-PAGE analysis revealed that cytosolic antigen is a mixture of polypeptides with molecular mass ranging from 27 to 96 KDa. The prominent polypeptides correspond to molecular weight 48 KDa and 38 KDa (Fig.15). 97 kD 66 kD 96kD 64kD 48kD 45 kD 38kD 31kD 29 kD Fig.15 SDS profiling of cytosolic proteins in comparative with ladder Enhancement of cell-mediated immunity A positive DTH response towards cytosolic antigen is an indication of cell-mediated immunity. As shown in (Fig.16), immunization with cytosolicantigen induced thehighest level of DTH. 10 days post immunization the DTH response in the group immunizedwith cytosolic antigen increased substantially followed by levels of DTH induced by PBS control BALB/c mice. Mice immunized with cytosolic antigen along with chitosan induced a higher DTH response when compared to antigen alone . DTH Fig.16 DTH responses in terms of footpad swelling following intra dermal inoculation of the antigen Cell proliferation assay by Trypan blue exclusion method Vaccine-induced stimulation of the cell-mediated immune response was further investigated through the capacity of spleen and lymph node cells to proliferate in response to concanavalin A and cytosolic antigen after immunization. The in vitro recall responses were evaluated 10 days after immunization. Cytoslic antigen induced strong proliferative in both cases (splenocytes and lymphocytes). This further indicates that cytosolic antigen is a potent stimulator of cell mediated immunity and the presence of chitosan as an adjuvant is not enhancing its immunogenicity (Fig.17) Fig.17 Lymphoproliferation responses after in vitro restimulation by cytosolic antigen evaluated by Trypan blue exclusion method NO estimation The nitrite concentration as an indicator of NO production by activated splenocytes was estimated in vitro for all the experimental groups. MaximumNO production was observed in Normal + ConA group followed by Ag+ Adj. (Fig.18). NO estimation for splenocytes Fig.18 NO estimation for splenocytes by Griess Reaction Conclusion Successful vaccination for the control of parasite multiplication is often related to antigen induced DTH response as an indication of activation of cell-mediated response. 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