Measurement of Acid Ecto-phosphatase Activity in Live Leishmania donovani Parasites

Materials and Reagents

1. Plastic cuvettes for spectrophotometric analysis (i.e., Sigma-Aldrich, catalog number: C5677) 
2. Plastic flasks for parasite culture 25 and 50 cm²
3. Plastic pipette tips (for 20 μl, 200 μl and 1 ml pipettes)
4. Sterile plastic pipettes (1 ml, 2 ml, 5 ml and 10 ml, from any company)
5. Falcon plastic tubes 15 and 50 ml (from any company)
6. Plastic Eppendorf tubes 1.5 ml (from any company)
7. Gilson or Eppendorf pipettes (or from any other company) for 1 μl, 20 μl, 200 μl and 1 ml volumes
8. L. tarentolae promastigotes (LEXSY host P10, Jena Biosciences, catalog number: LT-101)
9. pLEXSY-sat2 vector (Jena Biosciences, catalog number: EGE-274)
10. Brain Heart Infusion (Lexsy Broth BHI, Jena Biosciences, catalog number: ML-412S), stored at 4 °C when diluted in sterile dH₂O
11. Hemin (Jena Biosciences, catalog number: ML-108L), stored at RT 
12. L-biopterin (Cayman, catalog number: 10007662), stored at -20 °C
13. Nourseothricin (LEXSY NTC, Jena Bioscience, catalog number: AB-102L). Prepare solution of 10 mg/ml with sterile dH₂O, store in aliquots of 100 μl at -20 °C and after thawing at 4 °C
14. RPMI 1640-GlutaMax (Thermo Fisher, catalog number: 61870-010), stored at 4 °C 
15. Schneider’s (Sigma-Aldrich, catalog number: S-9895), stored at 4 °C 
16. hiFBS: FBS (From Gibco, Thermo Fisher, Biosera or any other company tested), stored at -20 °C, inactivated at 56 °C for 30 min before added to the culture medium
17. Penicillin-Streptomycin 10,000 U/ml (100x concentrated, Thermo Scientific, catalog number: 15140-122 or from any other company tested), stored at -20 °C
18. HEPES 1 M (Thermo Fisher Scientific, catalog number: 15630080), stored at 4 °C
19. 4-Nitrophenyl phosphate disodium salt hexahydrate, pNPP (Sigma-Aldrich, catalog number: P4744), powder stored at -20 °C. Prepare solution of 10 mM fresh for each experiment
20. 90 mM citrate buffer at pH 4.8 (25 °C) (Sigma-Aldrich, catalog number: C2488), stored at 2-8 °C
21. NaOH 0.5 M solution (Applichem, catalog number: 181692.1211), stored at RT 
22. Sodium tartrate (5 mM) solution; L(+)-tartaric acid (Merck, catalog number: 100804, or Sigma-Aldrich, catalog number: T109), stored at RT
23. 0.4% (w/v) Trypan Blue solution (Gibco, catalog number: 15250061; or Sigma-Aldrich, catalog number: T8154)
24. Formalin 100% (37% w/v formaldehyde solution; MERC: catalog number F8775 or from any other comany)
    

Equipment

1. Hemocytometer (Malassez, Marienfeld, catalog number: 0640610)
2. Incubator operating at 25 °C for the maintenance of parasite cultures (for example Shel-lab microbiological or CO₂ incubator, https://www.sheldonmanufacturing.com/shel-lab-incubators or from any other company)
3. Water bath (i.e., ED13, Julabo)
4. Analytical balance (i.e., ΑΕ50, Mettler) 
5. Spectrophotometer (i.e., SmartSpec Plus Spectrophotometer, 170-2525, Bio-Rad)
6. Tabletop centrifuge (i.e., Jouan CR3i multifunction, Thermo)
7. Microcentrifuge (i.e., Eppendorf 5417R)
   

Software

1. GraphPad Prism Software 5.01 (GraphPad, San Diego, CA)

Procedure

1. Leishmania promastigote axenic culture
   1. L. donovani, L. major or L. infantum promastigotes grow as suspension cells in axenic (in vitro cultures). 
   2. When starting from a frozen vial, thaw the cells quickly by immersing the vial in a beaker with water at 25 °C, transfer with a sterile 1 ml pippete in a falcon tube with 9 ml culture medium- [RPMI 1640-GlutaMax, supplemented with 10% (v/v) hiFBS, 40 mM HEPES and antibiotics (penicillin-streptomycin at 1x dilution (100 U/ml)], resuspend and centrifuge at 1000 x g for 7 min at RT.
   3. Remove the S/N, resuspend in 10 ml culture medium and transfer into a 25 cm² flask, close cap (without filter).
   4. Culture at 25 °C in the absence of CO₂ as described previously (Papadaki et al., 2015).
   5. Parasite subculturing is performed at 10⁶ parasites/ml and at the 6^th-7^th day of culture the parasite density is ~2 x 10⁷-2.5 x 10⁷ parasites/ml. 
   6. Parasite harvesting is performed by centrifugation (1,000 x g, 7 min at RT). 
   7. Parasites can be cultured for up to 10 passages and then a new culture should be started from a vial with frozen cells. 
   8. Culture L. tarentolae promastigotes at 25 °C in Brain Heart Infusion (Lexsy Broth BHI) medium supplemented with hemin (5 μg/ml) at a final concentration of 0.25% (w/v), 10 μM L-biopterin and penicillin-streptomycin (1x).
   9. Genetically modified parasites are cultured in similar conditions with the parental strains but with addition of Nourseothricin (100 μg/ml) when the genetic modification was performed with plasmids based on the pLEXSY-sat2 vector.
   10. For parasite maintenance, L. donovani strains(10⁷ cells) are subcultured once a week by removing 9 ml of the old culture and complementing with 9 ml of fresh medium (1/10 dilution).
   11. For cell counting
       
       1. emove a small volume of the culture (100 μl) for counting the cells with a hemocytometer (Malassez) and transfer it into an Eppendorf tube containing 900 μl 2% (w/v) formalin, at RT.
       2. Wait for 2-3 min.
       3. Take 100 μl and place in one chamber of the hemocytometer.
          Note: For use of hemocytometer, you can look at videos like https://www.youtube.com/watch?v=WWS9sZbGj6A.
          
       4. Observe under the microscope with a 40x magnification lens.
          
       5. For counting cells in the Mallassez hemocytometer, see image (Figure 1), below for a representation of the mallassez chamber.
          
       
       
       Figure 1. Graphical representation of the Mallassez hemocytometer chamber. Count the cells in several or in all the large squares (each square has an area of 0.05 mm² and 0.2 mm depth representing a volume of 0.01 mm³ = 10^-5 ml). Divide the number of cells counted by the number of squares observed and multiply by 10⁵/ml. Then multiply with the dilution factor (in this case by 10). If the parasite culture is less dense, dilute in a smaller volume of formalin before counting.
       
       
2. Protocol of assaying acid ecto-phosphatase in live Leishmania promastigotes
   The acid ecto-phosphatase activity at the surface of live Leishmania promastigotes, is assayed at 25 °C or 37 °C for 30 min with the phosphatase substrate p-nitrophenyl phosphate (pNPP) (10 mM) in a 90 mM citrate buffer at pH 4.8 (Sigma-Aldrich), with or without sodium tartrate (5 mM , L(+)-tartaric acid; Merck). Hydrolysis of the esteric bond and dephosphorylation of pNPP produces the p-nitro-phenol compound which after terminating the reaction by the addition of 0.5 Ν NaOH is quantified by measuring its absorbance at 405 nm (Figure 2).
   
   
   Figure 2. Diagram of the enzymatic reaction of dephosphorylation of the pNPP substrate. The acid ecto-phosphatase reaction at the surface of live Leishmania promastigotes can be carried out either at the logarithmic phase of growth (48-72 h post inoculation of the culture) or at the static phase of growth (6^th or 7^th day post-inoculation).
   
   
   1. Harvest Leishmania promastigotes (1-2 x 10⁷) by centrifugation (1,000 x g, 7 min, RT).
   2. Resuspend the cell pellet with 5 ml 100 mM HEPES solution and wash twice with the same buffer.
   3. Count cells (as in step 11, A) and assess cell viability by visual inspection under the microscope of the parasite motility (dispense 50 μl of cell suspension on a microscope slide and observe with a 4x lens) and quantitation by staining with 0.4% (w/v) Trypan Blue (i.e., as in Trypan Blue Staining protocol). Counting and cell viability could be performed simultaneously.
   4. Centrifuge the large volume of the cell suspension (1,000 x g, 7 min).
   5. Discard the supernatant (S/N). 
   6. Resuspend the cell pellet in 500 μl reaction solution containing the phosphatase substrate p-nitrophenyl phosphate (10 mM pNPP in a 90 mM citrate buffer, pH 4.8), with or without sodium tartrate 5 mM L(+)-tartaric acid prewarmed in the appropriate temperature of the reaction (25 °C or at 37 °C).
   7. Incubate cells in the reaction mixture at 25 °C or 37 °C for 30 min. 
   8. Terminate the reaction by the addition of 0.5 N NaOH (1 ml/ sample).
   9. Remove the parasites by centrifugation (2,000 x g, 5 min). 
   10. Transfer the S/N in a plastic cuvette for spectrophotometric analysis.
   11. Measure the absorbance of the reaction product (p-nitrophenolate = pNP) at 405 nm.
   12. Calculate the enzyme activity (A₄₀₅ values) and normalize for the mean number of cells/ml for each independent experiment (A₄₀₅/10⁷ cells/ml).
   13. Evaluate the acid phosphatase activity due to cell lysis under the reaction conditions as follows: 
       
       1. Incubate reaction buffer, equal volume to the phosphatase assay for each cell type, with the same number of Leishmania promastigotes used for each reaction, in the absence of substrate for 30 min, at 25 °C or 37 °C depending on the temperature you are performing the reaction.
       2. Recover the S/N after centrifuging down the cells (2,000 x g, 5 min).
       3. Add 10 mM pNPP.
       4. Incubate for 30 min.
          
       5. Measure acid phosphatase activity as described above. 
   14. Access cell viability as described in Step B2.
       

Data analysis

The acid phosphatase activity could be presented in a graph format (Figure 3) or a table (Table 1). Graphs and statistical significance were prepared and analyzed using GraphPad Prism Software 5.01 (GraphPad, San Diego, CA). The paired Student’s t-test was used to evaluate statistical significance among groups. A P value < 0.05 was considered statistically significant.


Figure 3. Graphical representation of the results of acid ecto-phosphatase assay in live promastigotes of four different Leishmania sp (L. donovani strain LG13, L. infantum strain GH12, L. major strain Friedlin, L. tarentolae strain LEXSY). The phosphatase activity was estimated in the presence of 5 mΜ sodium tartate (+ tartate) or in the absence of the inhibitor. Values are normalized to the % of live cells. A. The assay was performed without sodium tartate. B. 5 mM Sodium tartate was added to the assay mixture. The L. major-Friedlin strain had dramatically higher activity, ~7-8 fold, than the L. infantum-GH1 and L. donovani-LG13 strains, respectively, and this activity was > 90% sodium tartrate resistant. Error bars are Stds from 3 independent experiments. **, ***P ≤ 0.01 & ≤ 0.001 respectively, compared with corresponding control values (L. donovani-LG13), using a two-tailed paired Student’s t-test. ns: not significant.

Table 1. Acid ecto-phopshatase activity in live Leishmania spp promastigotes of the static phase of growth. Results are means from 3 independent experiments ± STD values *, **P ≤ 0.05 & 0.01, respectively, statistically significant differences as compared with control values (L. donovani strain LG13), two-tailed paired Student’s t-test. (Papadaki Amalia, PhD thesis 2015, https://www.didaktorika.gr/eadd/handle/10442/39638). Values are normalized to the % of live cells.

Notes

1. Viability of the cells should be assessed before and after incubation with the reaction medium qualitatively by visual inspection under the microscope of the parasite motility and quantitatively by 0.4% (w/v) Trypan Blue staining or by staining with another vital dye, as described in the protocol. 
2. Additionally, it should be confirmed that the enzymatic activity measured in the assay with live cells is due to acid ecto-phosphatase(s) and not to released enzymes by a) incubating the same number of parasites used in the assay with the reaction medium at 37 °C for 30 min, b) removing the parasites by centrifugation and c) testing for pNPP hydrolysis in this medium devoid of cells (Cedro Fernandes et al., 2003).
   

Acknowledgments

This work was funded by the Hellenic Pasteur Institute (http://www.pasteur.gr) AP and HB, a sponsorship from Genesis Pharma S.A (http://www.genesispharma.gr/homeeng.htm) HB, and a Bilateral Research & Technology Collaboration Greece-France 2013 grant (No. 1811) funded by the Greek General Secretariat for Research and Technology (http://www.gsrt.gr/central.aspx?sId=119I428I1089I323I488743) AP and HB.

Competing interests

The authors have no financial or non-financial competing interests on the information provided in this manuscript.

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