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May 2015

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Measurement of Acid Ecto-phosphatase Activity in Live Leishmania donovani Parasites
杜氏利什曼原虫中酸性胞外磷酸酶活性的活体检测   

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Abstract

Acid ecto-phosphatases are enzymes that hydrolyze phosphomonoesters in the acidic pH range with their active sites facing the extacellular medium. Their activities can be measured in living cells. In bacteria and protozoan pathogens, acid ecto-phosphatases have been associated with the survival of intracellular pathogens within phagocytes through inhibition of the respiratory burst, suggesting that they act as virulence factors. Extracellular acid phosphatase activity in Leishmania (L.) donovani has been associated with the degree of promastigote virulence/infectivity. The levels of acid ecto-phosphatase activity in different Leishmania sp or even strains of the same species vary and this has been linked to their virulence. It may also be related to their ability to survive and multiply in the insect host.

Acid phosphatase enzymatic activity can be measured in crude membrane fractions and in membrane fractions enriched in plasma membrane, however, in these cases, the intracellular acid phosphatases, mainly localized in lysosomes, contribute to the final result. Therefore, measuring phosphatase activity at the surface of live cells in acidic pH range is the only accurate way to measure acid ecto-phosphatase activity. This assay is performed at 25 °C or 37 °C for 30 min using as substrate the generic phosphatase substrate p-nitrophenyl phosphate (pNPP), in a citrate buffer, with or without sodium tartrate (L(+)-tartaric acid), as histidine acid phosphatases are classified according to their sensitivity to tartate inhibition. The steps of the protocol consist of pelleting cells in suspension, in this case Leishmania promastigotes, washing twice with HEPES buffer, resuspending the cells in the substrate reaction mixture and terminating the reaction by the addition of 0.5 N NaOH. The cells are removed by centrifugation and the absorbance of the reaction product (p-nitrophenolate=pNP) in the supernatant is measured at 405 nm. The enzymatic activity (A405 values) is normalized for the mean number of cells/ml used for each independent experiment.

Keywords: Ecto-enzymes (胞外酶), Acid ecto-phosphatases (酸性胞外磷酸酶), Enzymatic assay in live cells (活细胞酶活分析), Leishmania sp (利什曼原虫), phosphatase activity (磷酸酶活性 ), Tartate sensitivity (酒石酸盐敏感性 )

Background

Trypanosomatid protozoan parasites are a diverse group of primitive parasitic organisms (Kaufer et al., 2017). They are both of medical and veterinary importance as many Trypanosomatid species cause serious or fatal diseases in humans and agricultural animals in tropical and subtropical regions worldwide (Reference 16). Virulence factors of these parasites include molecules expressed on their cell surface as well as those secreted or shed into the extracellular medium (Gottlieb and Dwyer, 1981; Fonseca-de-Souza et al., 2008 and 2009). Trypanosomatidae are exposed to diverse environmental stimuli throughout their life cycles in all biological systems they reside. Central events in recognition of the external and internal signals in these cells, which lead to specific responses for adaptation and survival in all these environments, are protein phosphorylation and dephosphorylation events carried out by a multitude of kinases and phosphatases. The trypanosomatid kinome and phosphatome have been recently deciphered (Brenchley et al., 2007; Sadatomi et al., 2013).

Phosphatase activities modulate different aspects of the infection by members of the trypanosomatid family (Neves et al., 2014). Ecto-phosphatases, the enzymes localized in parasite’s surface membranes, are of special interest as they are candidate molecules for the interaction between the parasite and its host and thereby they could be intimately involved in many cellular events implicated in parasite adhesion to the host, protection from the host’s immune response and establishment of infection. Moreover, several alternative functions have been suggested for ecto-phosphatases, such as participation in proliferation, differentiation, adhesion, virulence and infection (Katakura and Kobayashi, 1988; Singla et al., 1992). Little is known about their physiological roles in protozoa parasites (Cosentino-Gomes et al., 2011; Gomes et al., 2011). In particular, acid ecto-phosphatases that hydrolyze phosphomonoesters at acidic pHs, have been associated with the survival of intracellular parasites within phagocytes through inhibition of the respiratory burst (Saha et al., 1985; Remaley et al., 1985; Baca et al., 1993; Reilly et al., 1996; Jungnitz et al., 1998; Saleh and Belisle, 2000; Burtnick et al., 2001; Steenkamp, 2002; Cosentino-Gomes et al., 2009; Aragon et al., 2011; Cosentino-Gomes and Meyer-Fernandes, 2011), suggesting that they may act as virulence factors (Katakura and Kobayashi, 1988; Singla et al., 1992; Papadaki et al., 2015). They may also be related to the ability of parasites to survive and multiply in the insect host (Remaley et al., 1985; Saha et al., 1985; Singla et al., 1992). The field of the acid ecto-phosphatases was reopened with the characterization of the L. donovani acid ecto-phosphatase LdMAcP (Papadaki et al., 2015) and the identification of at least two more open reading frames in the L. donovani genome that could be potentially expressed at the surface membrane of the parasites (Papadaki and Boleti. data not published).

Therefore assaying acid phosphate activity at the surface of live cells is a putative diagnostic parameter for virulence of Leishmania parasite strains as well as for the evaluation of the expression of the ecto-phosphatases under study at the surface of transgenic parasite strains overexpressing them.Acid phosphatase enzymatic activity can be measured in crude membrane fractions and in membrane fractions enriched in plasma membrane (Glew et al., 1982; Shakarian et al., 2002), however, in these cases, the intracellular acid phosphatases, mainly localized in lysosomes, contribute to the final result.

The protocol provided herein was used to measure acid ecto-phosphatase activity in live Leishmania promastigotes but it could be used to detect and quantitate acid ecto-phosphatase activity in all suspension cells of prokaryotic or eukaryotic origin.

Our method was adapted from previously described methodologies (Remaley et al., 1985; Saha et al., 1985; Katakura and Kobayashi, 1988; Singla et al., 1992) for assaying ecto-phosphatases in other organisms.

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 6th-7th 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 (6th or 7th 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.

References

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简介

酸性磷酸酶是在酸性pH范围内水解磷酸单酯的酶,其活性部位面向外细胞培养基。它们的活动可以在活细胞中测量。在细菌和原生动物病原体中,酸性E-磷酸酶通过抑制呼吸爆发而与吞噬细胞内的细胞内病原体的存活相关,表明它们作为毒力因子。E.> Leishmania(L.)Doovayi >胞外酸性磷酸酶活性与前鞭毛体毒力/感染程度有关。同一品种的Leishmania、> > SP、甚至同一菌株的酸性磷酸酶活性不同,这与它们的毒力有关。这也可能与它们在寄主昆虫中的生存和繁殖能力有关。



酸性磷酸酶的酶活性可以在粗膜组分和富含膜质膜组分中进行测定,然而,在这些情况下,主要定位于溶酶体中的细胞内酸性磷酸酶有助于最终结果。因此,在酸性ph范围内测定活细胞表面磷酸酶活性是测定酸性外磷酸酶活性的唯一准确方法。本试验在25°C或37°C下进行,30分钟,使用作为底物的通用磷酸酶底物对硝基苯基磷酸酶(p>npp),在柠檬酸缓冲液中,加入或不加入酒石酸钠(L+-酒石酸),作为组氨酸磷酸酶,根据其对酒石酸抑制的敏感性进行分类。该方案的步骤包括悬浮液中的造粒细胞,在这种情况下,EM>Leishmania>前鞭毛体,用HEPES缓冲液洗涤两次,将细胞重新悬浮在底物反应混合物中,并通过加入0.5 N NaOH终止反应。通过离心去除细胞,并在405 nm处测量反应产物(p>-硝基苯酚=p>np)在上清液中的吸光度。酶活性(A<>405</SUB>值)归一化为每个独立实验所用的平均细胞数/ml。
【背景】锥虫原生动物寄生虫是一组多样的原始寄生生物(Kaufer等人,2017年)。它们都是医学和兽医的重要性,因为许多锥虫类物种在热带和亚热带地区的人类和农业动物中引起严重或致命的疾病(参考文献16)。这些寄生虫的毒力因子包括在细胞表面上表达的分子以及分泌或脱落进入细胞外基质的分子(戈特利布和DeYER,1981;Fonseca de Souza 等。,2008和2009)。锥虫在它们所居住的所有生物系统中,在其生命周期中受到不同的环境刺激。识别这些细胞中的外部信号和内部信号的中心事件,导致在所有这些环境中适应和存活的特定反应,是由大量激酶和磷酸酶所进行的蛋白质磷酸化和去磷酸化事件。最近发现了锥虫动蛋白和磷光体(Brenchley 等, >,2007;Sadatomi 等。,2013)。



磷酸酶活性调节锥虫体家族成员感染的不同方面(Neves等人,2014年)。在疟原虫表面膜中定位的酶是特别有趣的,因为它们是寄生虫和宿主之间相互作用的候选分子,因此它们可以紧密地参与寄生虫附着到宿主的许多细胞事件,保护宿主免受免疫反应。感染的建立。此外,已经提出了几种可替代的功能,如参与增殖、分化、粘附、毒力和感染(Kakula和Kabayayi,1988;SunLaEm>等, >,1992)。人们对它们在原生动物寄生虫中的生理作用知之甚少(Cosentino Gomes等人,2011;Gomes等人,2011)。特别是在酸性PHS中水解磷酸单酯的酸性外膜磷酸酶,通过抑制呼吸爆发而与吞噬细胞内的寄生虫存活有关(Saha 等< ,1985;Remaley 等< ,1985;Baa> Em>等。,1993;蕾莉等。 >,1996;Jungnitz 等.>1998;萨利赫和BelISle,2000;Burtnick 等。>2001;Stun KAMP,2002;Cosentino Gomes 等。 2009;阿拉贡等。,2011;Cosentino Gomes和Meyer Fernandes,2011),表明它们可能作为毒力因子(Kakula and Kabayasi,1988;SunLa等。 1992;PAPADAI 等. ,2015)。它们也可能与寄生虫在昆虫宿主中生存和繁殖的能力有关(remaley等,1985;saha等,1985;singla等,1992)。用E.> L. donovani 酸性磷酸酶 LD > MACP(PAPADAKI 等)< ,对酸性磷酸酶的活性场进行重新开放。2015)和在 L. donovani >基因组中的至少两个开放阅读框的鉴定,该基因可能在寄生虫的表面膜上表达(PAPADAKI和BelETI)。数据未发布)。



因此,测定活细胞表面的酸性磷酸酶活性是Em>Leishmania<E/E>寄生虫菌株的毒力的一个重要的诊断参数,同时也可用于研究过度表达的转基因寄生虫菌株表面的Et-磷酸酶的表达。可在粗膜组分和富集在质膜中的膜组分(格鲁等,>1982,SkaRial[Em>et al.2002)]进行测定,但在这些情况下,主要定位于溶酶体中的细胞内酸性磷酸酶有助于最终结果。



本文提供的方法用于测定活 Leishmania 前鞭毛体中的酸性磷酸酶活性,但它可用于检测和定量原核或真核来源的所有悬浮细胞中的酸性磷酸酶活性。



我们的方法适用于先前描述的方法(Remaley 等.,1985;Saha 等.,1985;Kakula和Kabayasi,1988;SunLa等< ,1992),用于测定其他生物体中的ECT磷酸酶。

关键字:胞外酶, 酸性胞外磷酸酶, 活细胞酶活分析, 利什曼原虫, 磷酸酶活性 , 酒石酸盐敏感性

材料和试剂

  1. 分光光度分析用塑料比色杯(即>,Sigma-Aldrich,目录号:C5677)
  2. 寄生虫培养用塑料瓶25和50 cm2
  3. 塑料吸管头(适用于20μl、200μl和1 ml吸管)
  4. 无菌塑料移液管(1毫升、2毫升、5毫升和10毫升,任何公司提供)
  5. Falcon塑料管15和50毫升(来自任何公司)
  6. 塑料Eppendorf试管1.5毫升(任何公司提供)
  7. Gilson或Eppendorf移液管(或任何其他公司的移液管),容量分别为1μl、20μl、200μl和1 ml
  8. E.> TrutoLoa>前鞭毛体(LosiSH P10,耶拿生物科学,目录号:LT-101)
  9. Plexsy-Sat2载体(Jena Biosciences,目录号:EGE-274)
  10. 脑心灌注(LISSY肉汤BHI,耶拿生物科学,目录号:ML412S),储存于4℃时稀释在无菌DH<2 < /Su>
  11. Hemin(Jena Biosciences,目录号:ML-108L),储存于RT
  12. L-生物喋呤(开曼群岛,目录号:10007662),储存于-20°C
  13. 牛血清蓖麻素(Lexsy NTC,Jena Bioscience,目录号:AB-102L)。用无菌dh2o制备10mg/ml溶液,在-20℃下分100μl保存,在4℃解冻后保存。
  14. RPMI 1640谷氨酰胺(Thermo Fisher,目录号:61870-010),储存于4°C下
  15. 施耐德(Sigma-Aldrich,目录号:S-9895),储存于4°C
  16. HIFBS:FBS(从GiBCO、TeaMiFisher、BioSeaR或其他任何公司测试),储存在-C°C,在56℃下灭活30分钟,然后加入培养基中。
  17. 青霉素链霉素10000 U/ml(100倍浓缩,热科学,目录号:15140-122或来自任何其他公司测试),储存于-20°C
  18. HEPES 1 M(Thermo Fisher Scientific,目录号:15630080),储存于4°C
  19. 4-硝基苯基磷酸酯二钠盐六水合物,p>npp(sigma aldrich,目录号:p4744),粉末储存在-20°C下。为每个实验准备10 mm新鲜溶液
  20. pH 4.8(25°C)的90 mm柠檬酸盐缓冲液(Sigma-Aldrich,目录号:C2488),储存于2-8°C
  21. NaOH 0.5 M溶液(APPLICHEM,目录号:181692.1211),储存于室温下
  22. 酒石酸钠(5 mm)溶液;L(+)-酒石酸(默克,目录号:100804,或西格玛·奥尔德里奇,目录号:T109),储存在室温下
  23. 0.4%(w/v)台盼蓝溶液(Gibco,目录号:15250061;或Sigma-Aldrich,目录号:T8154)
  24. 福尔马林100%(37% W/V甲醛溶液;MEC:目录号F875或任何其他CONMANE)< BR/>

设备

  1. 血细胞仪(Malassez,Marienfeld,目录号:0640610)
  2. 在25°C下运行的用于培养寄生虫培养物的孵化器(例如,Sel-Lab微生物或CuSub>2</Sub孵育器,< HREF=“http://www-SeldOnguto.Com/Sel-Lab孵器”目标=“空白”> http://www. SeldoNeald.com /sEL实验室孵化器或来自任何其他公司)
  3. 水浴(即,ed13,julabo)
  4. 分析天平(>>50,梅特勒);
  5. Spectrophotometer(即>,StaseSpple Plus分光光度计,170—2525,BIO RAD)
  6. 台式离心机(即,Juang-CR3i多功能,热)
  7. Microcentrifuge(即>,埃彭多夫54 17R)< BR>

软件

  • GraphPad Prism软件5.01(GraphPad,加利福尼亚州圣地亚哥)
  • 程序

    1. 利什曼原虫原鞭毛体无菌培养
      1. Em>L. donovani>EM>>Em>EM>EM>EM>L.
      2. 当从一个冰冻的小瓶开始时,用25℃的水将烧杯浸入烧杯中,然后用1毫升的无菌培养液在9毫升培养基中用100毫升的培养液(1640,40)(10%)和抗生素(青霉素稀释1倍稀释)(50毫升),在室温下1000x g下再悬浮和离心7分钟。
      3. 取出序列号,在10毫升培养基中重新培养,并转移到25厘米的2烧瓶中,盖上盖子(不带过滤器)。
      4. 在25°C的培养中,没有Co<2>/Sub>(PAPADAKI等2015)。
      5. 寄生虫继代培养于10 6 寄生虫/ml,在6 -7(SUP> TH<SUP/SUP>培养基中,寄生虫密度为~2×10)SUP> 7 2.5×10 10,SUP> 7 寄生虫/ml。
      6. 寄生虫采集通过离心(1000x g,rt下7分钟)进行。
      7. 寄生虫可以培养多达10个通道,然后一个新的文化应从一个小瓶与冷冻细胞。
      8. 培养液>E.> TalutoLe > 25℃脑灌流(Listy Burth-BHI)培养基,终浓度为0.25%(W/V)、10μm L生物蝶呤和青霉素链霉素(1X),添加hemin(5μg/ml)。
      9. 基因修饰的寄生虫在与亲本菌株相似的条件下培养,但在使用基于Plexsy-Sat2载体的质粒进行基因修饰时添加牛血清蓖麻素(100μg/ml)。
      10. 对于寄生虫的维持,Em> L. donovani 菌株(10×SUP> 7细胞),每周除去一次,除去9毫升的旧培养物,再加入9毫升新鲜培养基(1/10稀释液)。
      11. 对于细胞计数
        1. 用血球计(Malasez)计数小细胞培养物(100μL),并将其转移到含有900μl 2%(W/V)福尔马林的EpBordRF管中,在RT.。
        2. 等待2-3分钟。
        3. 取100μl,置于血细胞仪的一个室中。
          注意:使用血细胞仪时,您可以观看像>https://www.youtube.com/watch?V=WWS9SZBGJ6A>。> < BR>
        4. 用40倍放大镜在显微镜下观察。
        5. 为了计算Malasez血球计中的细胞,请参阅图像(图1),下面是Malasez腔的表示。< BR>
        < BR> < IMG类=“DoOtStWh”宽度=“300”高度=“190”ALT=“”SRC=“/附着/图像/ 20190928 /20199280845 52Y7172.JPG”/>
        图1。Malasez血球计室的图形表示。< /强>计数在几个或所有大方格中的细胞(每个方块的面积为0.05毫米 2和0.2毫米深度代表0.01毫米的体积 3 =10 5 ml)。将计数的细胞数除以观察到的平方数,再乘以105/ml。然后乘以稀释因子(在本例中为10)。如果寄生虫培养密度较低,则在计数前用较小体积的福尔马林稀释。< BR> < BR>
    2. 利什曼原虫前鞭毛体酸性外磷酸酶测定方法的研究 用磷酸酶底物对硝基苯基磷酸酯(p>npp)(10 mm)在pH 4.8(Sigma-Aldrich)的90 mm柠檬酸缓冲液中,在25°C或37°C下测定活利什曼原虫>前鞭毛体表面的酸性外磷酸酶活性30分钟,并加入或不加入酒石酸钠(5 mm,L(+)-酒石酸;默克)。酯化键的水解和p>npp的去磷酸化产生对硝基苯酚化合物,在通过添加0.5_naoh终止反应后,通过测量其在405nm处的吸光度来量化该化合物(图2)。
      < BR> < IMG类=“DoOtStWh”宽度=“300”高度=“165”ALT=“”SRC=“/附着/图像/ 20190928 /20199280846153322.JPG”/>
      图2。Em>P> NPP底物的去磷酸化反应的示意图。< <强>>Leishmania < 前鞭毛体的酸性磷酸酶反应可在生长的对数生长期(培养后48~72小时)或生长静止期(6 Th)进行。接种后第7天)。 < BR>
      1. 用离心法(1000x g,7分钟,rt)采集利什曼原虫前鞭毛体(1-2x 107)。
      2. 用5毫升100毫米HEPES溶液重新悬浮细胞颗粒,并用相同的缓冲液洗涤两次。
      3. 计数细胞(如步骤11,A),通过显微镜下观察寄生虫活力(在显微镜载片上分配50μl细胞悬液并用4X透镜观察)评估细胞活力,并用0.4%(W/V)台盼蓝染色(即>)进行定量。如在台盼蓝染色议定书。计数和细胞存活率可以同时进行。
      4. 离心大体积细胞悬液(1000x g,7分钟)。
      5. 丢弃上清液(序列号)。
      6. 在90 mL柠檬酸盐缓冲液中,在pH为4.8的柠檬酸盐缓冲液中,在500μL反应溶液中回收细胞颗粒,在90℃柠檬酸盐缓冲液(pH 4.8)下,加入或不加入酒石酸钠5 mL L(+)-酒石酸,在适当的反应温度(25℃或37℃)下进行预处理。
      7. 将细胞在25°C或37°C的反应混合物中培养30分钟。
      8. 通过加入0.5 N NaOH(1毫升/样品)终止反应。
      9. 离心(2000x g>,5分钟)除去寄生虫。
      10. 将序列号转移到塑料试管中进行分光光度分析。
      11. 在405nm处测量反应产物的吸光度(p>-硝基苯酚=p>np)。
      12. 计算酶活性(A<<405</SUB>值),对每个独立实验(A<405<405分/次>7>7 /SUP>细胞/ml)的平均细胞数/ml进行归一化处理。
      13. 在反应条件下评价细胞裂解引起的酸性磷酸酶活性:
        1. 孵育反应缓冲液,与每个细胞类型的磷酸酯酶体积相等,与每个反应所用的Leishmania 前鞭毛体相同数目,在底物30分钟、25℃或37℃的情况下,取决于你们进行反应的温度。
        2. 离心细胞(2000x g,5min)后恢复s/n。
        3. 增加10 mmpnpp。
        4. 孵育30分钟。
        5. 如上所述测量酸性磷酸酶活性。
      14. 如步骤b2所述接入小区生存能力。

    数据分析

    酸性磷酸酶活性可以用图表形式(图3)或表格(表1)表示。使用GrAPPAD棱镜软件5.01(GrAPPad,圣地亚哥,CA)准备和分析图表和统计意义。配对学生的 t>检验,评价各组间的统计学意义。AP>值>0.05,差异有统计学意义。
    < BR> < IMG类=“DoOtStWh”宽度=“300”高度=“164”ALT=“”SRC=“/附着/图像/ 20190928 /201992808464 71.1453. JPG”/>
    图3。四种不同Leishmania sp活鞭毛体酸性磷酸酶测定结果的图解( L. donovani 菌株LG13, L.在5米塔酸钠(+酒石酸盐)或不存在抑制剂的情况下,估计了磷酸酶活性。值被归一化到活细胞的百分比。a.在没有酒石酸钠的情况下进行分析。b.向分析混合物中添加5 mm酒石酸钠。< L.Eng/EM> FreLDLIN菌株的活性显著高于L.GH1和L. donovani > LG13菌株,活性为Gt;90%酒石酸钠抗性。误差线是3个独立实验的标准差分。采用双尾配对的 t>检验,与对照值(L. donovani > -LG13)相比,*> <>>0.01和≤0.001。ns:不显著。
    < BR> 表1。生长期静止期利什曼原虫前鞭毛体的酸性磷酸酶活性< < /强>结果为3个独立实验,±STD值*,* P>≤0.05和0.01,与对照值( L. donovani >菌株LG13)比较,差异有统计学意义,双尾配对学生t检验。(Papadaki Amalia,博士论文2015,< HeRF= =“http://www-diktutkk.Gr/EdAd/Heale/1042/3638”目标=“yBLASH”>http://www-diktutik.Gr/EADAD/句柄/ 10442/39638</a>。值被归一化到活细胞的百分比。 < IMG类=“DoOtStWh”IG=“”宽度=“650”高度=“212”ALT=“”SRC=“/附着/图像/ 20190928 /20199280848 185516.PNG”/>

    笔记

    1. 细胞的活力应在培养基孵育前和孵育后定性评估,通过显微镜下观察寄生虫的活动性,并用0.4%(W/V)台盼蓝染色或用另一种重要染料染色,如协议中所述。
    2. 此外,应该确认,在活细胞分析中测定的酶活性是由于酸性E-磷酸酶(S)而不是由A释放的酶)在反应中培养相同数量的寄生虫,在37°C下用30分钟进行反应,b)离心去除寄生虫,C)在没有细胞的培养基中进行PNPP水解试验(Cedro Fernandes 等.,2003)。

    致谢

    这项工作是由希腊巴斯德研究所资助的(A HRFF=“http://www. PaSTur.G/[目标] =“http://www.巴斯德.Gr ”AP和HB,来源于GoEngEngA A的赞助(< HRFF=http://www. Geesistura.Gr/HOMENG.HTM)目标=“http:http://www. GunesiasPaa.Gr/HangEn.HTM )Hb,和一个双边研究和。技术合作希腊-法国2013赠款(第1811号)由希腊总研究和技术秘书处资助(“HREF=”HTTP//www. gSr.Gr/Calimal.ASPX?sid=119i428i1089i323i488743“target=”\u blank“>http://www.gsrt.gr/central.aspx?SID=119I428I1089I323I488743)AP和HB。

    竞争利益

    作者对本稿件所提供的信息没有财务或非金融利益。

    工具书类

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    引用:Papadaki, A. and Boleti, H. (2019). Measurement of Acid Ecto-phosphatase Activity in Live Leishmania donovani Parasites. Bio-protocol 9(19): e3384. DOI: 10.21769/BioProtoc.3384.
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