TITLE: Purification of germination-competent E. intestinalis spores
AUTHORS: Noelle V. Antao, Mahrukh Usmani, Frederick Rubino, Harshita Ramchandani, Pattana Jaroenlak, Kacie L. McCarty, Damian C. Ekiert, Gira Bhabha

[DESCRIPTION]
Encephalitozoon intestinalis is a human-infecting microsporidian species, which can cause fatal illnesses in immunocompromised patients. As an obligate intracellular parasite, E. intestinalis replicates exclusively within host cells, and then is transmitted via a non-replicating spore. Studying spores requires culturing the parasite within host cells, initially yielding a mixture of parasite stages, including both germination-competent and germination-incompetent spores. Starting from previously described protocols1–4, here, we describe an optimized method to reproducibly enrich and isolate mature, germination-competent E. intestinalis spores from infected Vero cells. We provide characterization of germination rates, infectivity as assessed using a parasite FISH staining protocol5  and genomic content of germination-competent spores. 


[STEPS]
SECTION: Section 1: Parasite propagation and host cell lysis
1. Seed 2.1 × 106 Vero cells in a T-75 tissue culture flask with DMEM:HG supplemented with 10% FBS and 1X Non Essential Amino Acids.
SECTION: Section 1: Parasite propagation and host cell lysis
2. After 48 hours, change media to DMEM: HG supplemented with 3% FBS and 1X Non Essential Amino Acids. Total volume of media in the flask should be 30 mL.
SECTION: Section 1: Parasite propagation and host cell lysis
3. Add E. intestinalis spores to the Vero cells at a multiplicity of infection (MOI) of 10.
SECTION: Section 1: Parasite propagation and host cell lysis
4. Replace the media (DMEM: HG supplemented with 3% FBS and 1X Non Essential Amino Acids) every 48 hours over the next 7 days.

 

SECTION: Section 1: Parasite propagation and host cell lysis
5. Using a cell scraper (30 mm), detach infected cells from the base of the flask.
SECTION: Section 1: Parasite propagation and host cell lysis
6. Transfer the flask contents to a 50 mL conical tube.
SECTION: Section 1: Parasite propagation and host cell lysis
7. Centrifuge the 50 mL conical tube at 2000 x g for 10 min at room temperature (RT).
SECTION: Section 1: Parasite propagation and host cell lysis
8. Remove and discard the supernatant.
SECTION: Section 1: Parasite propagation and host cell lysis
9. Resuspend the cell pellet in 12 mL of pre-chilled sterile Phosphate buffered saline (PBS) + 0.1% SDS.

 

SECTION: Section 1: Parasite propagation and host cell lysis
11. Vortex at max speed for 5 seconds.
SECTION: Section 1: Parasite propagation and host cell lysis
10. Incubate on ice for 5 minutes.
SECTION: Section 1: Parasite propagation and host cell lysis
13. Vortex at maximum speed for 5 seconds.
SECTION: Section 1: Parasite propagation and host cell lysis
12. Incubate on ice for 5 minutes.
SECTION: Section 1: Parasite propagation and host cell lysis
14. Divide the cell suspension equally into two 15 mL tubes (6 mL per tube).
17. Centrifuge at 4000 x g for 30 minutes at RT.
16. Mix the contents of the 15 mL tube well by inverting the tube until homogenous, followed by vortexing at maximum speed for 5 seconds.
18. Remove and discard the supernatant.
19. Resuspend the spore pellet in 10 mL PBS and transfer to a new 15mL tube. This is wash 1.
23. Resuspend the pellet in 1 mL of PBS and transfer to a 1.5 mL tube.
22. Repeat steps 19-21. This is wash 2.
21. Remove and discard the supernatant.
20. Centrifuge at 4000 x g for 10 minutes at RT.
SECTION: Section 3: Enrichment of germination-competent spores using a discontinuous Percoll gradient
27. Prepare a discontinuous Percoll gradient in an ultracentrifuge tube. Pipet 2.5 mL of each solution in the following order into the ultracentrifuge tube: 100% Percoll; 75% Percoll; 50% Percoll; 25% Percoll. Pipet each Percoll solution carefully along the wall of the tube, taking care not to disturb the layers. After pipetting is complete, all layers should be well defined and clearly visible.

 

25. Remove and discard the supernatant. Resuspend pellet in 1.5 mL PBS. We refer to this fraction as Crude spores.
24. Centrifuge at 4000 x g for 5 minutes at RT. This is wash 3.
SECTION: Section 3: Enrichment of germination-competent spores using a discontinuous Percoll gradient
28. Add the resuspended Crude spore pellet purified from the continuous Percoll gradient to the top of the discontinuous gradient.

 

SECTION: Section 3: Enrichment of germination-competent spores using a discontinuous Percoll gradient
38. Discard the supernatant and resuspend the pellet in fresh 1 mL PBS. We refer to this fraction as Purified spores.
SECTION: Section 3: Enrichment of germination-competent spores using a discontinuous Percoll gradient
37. Centrifuge at 4000 x g for 5 minutes. This is wash 2.
SECTION: Section 3: Enrichment of germination-competent spores using a discontinuous Percoll gradient
36. Transfer the resuspended pellet to a 1.5 mL tube.
SECTION: Section 3: Enrichment of germination-competent spores using a discontinuous Percoll gradient
35. Discard the supernatant and resuspend the pellet in 1 mL PBS.
SECTION: Section 3: Enrichment of germination-competent spores using a discontinuous Percoll gradient
34. Centrifuge at 12,700 x g for 10 minutes at RT. This is wash 1.
SECTION: Section 3: Enrichment of germination-competent spores using a discontinuous Percoll gradient
33. Resuspend the pellet in 1 mL PBS and transfer to a new ultracentrifuge tube containing 9 mL PBS and mix by pipetting.
SECTION: Section 3: Enrichment of germination-competent spores using a discontinuous Percoll gradient
32. Remove and discard all the contents of the tube, leaving behind ~0.5 mL containing the pellet fraction at the bottom of the tube.

 

SECTION: Section 3: Enrichment of germination-competent spores using a discontinuous Percoll gradient
31. After centrifugation, the spores will separate into two bands and a pellet. Band 1: Between the 75% and 50% Percoll zones, Band 2: Between the 100% and 75% Percoll zones, Pellet: Bottom of the tube.
 

SECTION: Section 3: Enrichment of germination-competent spores using a discontinuous Percoll gradient
30. Centrifuge at 12,700 x g for 30 minutes at RT.
SECTION: Section 3: Enrichment of germination-competent spores using a discontinuous Percoll gradient
29. Place tubes in a swinging bucket rotor.
SECTION: Section 2: Crude spore purification using Percoll
15. Add an equal volume (6 mL) of Percoll to each 15 mL tube containing the cell suspension.
SECTION: Section 3: Enrichment of germination-competent spores using a discontinuous Percoll gradient
26. Prepare Percoll solutions by diluting 100% Percoll in sterile PBS. Prepare the following percentages of Percoll solutions: 100% Percoll; 75% Percoll; 50% Percoll; and 25% Percoll. Mix dilutions by vortexing until homogeneous.
SECTION: Section 5: Measurement of infectivity in Vero cells
58. Add 100 μl of dye staining solution (Calcofluor white (2 μg/mL) and DRAQ5 (1:200) in PBS-T (0.1% Tween-20)).
SECTION: Section 5: Measurement of infectivity in Vero cells
57. Add 500 μl FISH wash buffer to each coverslip and incubate at 37 °C for 30 minutes. Protect samples from light by covering the humidity chamber with aluminium foil. This is wash step 2.

 

SECTION: Section 5: Measurement of infectivity in Vero cells
56. Aspirate FISH wash buffer.
SECTION: Section 5: Measurement of infectivity in Vero cells
55. Add 500 μl FISH wash buffer to each coverslip and incubate at 37 °C for 30 minutes. Protect samples from light by covering the humidity chamber with aluminium foil. This is wash step 1.
SECTION: Section 5: Measurement of infectivity in Vero cells
54. Aspirate FISH staining solution.
SECTION: Section 5: Measurement of infectivity in Vero cells
53. Incubate at 37 °C overnight. Protect samples from light by covering the humidity chamber with aluminium foil.
SECTION: Section 5: Measurement of infectivity in Vero cells
52. Add 50 μl FISH staining solution (125 nM FISH probe in hybridization buffer).
SECTION: Section 5: Measurement of infectivity in Vero cells
51. Wash coverslips with 500 μl FISH hybridization buffer.
SECTION: Section 5: Measurement of infectivity in Vero cells
50. Transfer coverslips to a humidity chamber.
SECTION: Section 5: Measurement of infectivity in Vero cells
47. After 24 hours, gently aspirate media without disturbing the cells, and wash cells with 2 mL PBS.
SECTION: Section 4: Measurement of germination rates
44. To quantify germination rates, count at least 100 spores across multiple fields of view.

 
    
 

SECTION: Section 4: Measurement of germination rates
43. Apply 4 μl of the germinated spore mixtures to poly-lysine coated slides. Cover with a square coverslip and image samples on a light microscope with a 60X or 63X objective under brightfield illumination. Polar tube germination can be visualized by phase contrast microscopy or DIC microscopy.
SECTION: Section 4: Measurement of germination rates
42. Incubate at 37 °C for 15 minutes.
SECTION: Section 4: Measurement of germination rates
41. Add 10 μl of germination buffer to each tube and mix by pipetting gently.
SECTION: Section 3: Enrichment of germination-competent spores using a discontinuous Percoll gradient
39. Store the tube from step 38 at 4 °C until further use. For E. intestinalis spore preparations, we typically observe minimal decline in germination rates over the course of several weeks. For other microsporidian species, this may vary, and the optimal storage solution may also vary.
SECTION: Section 4: Measurement of germination rates
40. Pipet 2 μl of spores purified in Step 38 (Purified spores, Band 1 and Band 2 spores) and Crude spores into 1.5 mL tubes.
SECTION: Section 5: Measurement of infectivity in Vero cells
49. Aspirate fixative and wash cells with 2 mL of PBS-T (0.1% Tween 20).
SECTION: Section 5: Measurement of infectivity in Vero cells
48. Add 2 mL of fixative (4% PFA in PBS-T (0.1% Tween 20)) and incubate at RT for 45 minutes.
SECTION: Section 5: Measurement of infectivity in Vero cells
46. To infect cells, add Crude spores, Purified spores, Band 1 and Band 2 spores at an MOI of 30.
SECTION: Section 5: Measurement of infectivity in Vero cells
45. Seed 2 × 105 Vero cells per well in a 6 well tissue culture plate. Cells should be cultured in DMEM:HG supplemented with 3% FBS and 1X Non Essential Amino Acids. Each well should have five 12 mm coverslips.

 

SECTION: Section 5: Measurement of infectivity in Vero cells
59. Incubate at RT for 30 minutes. Protect samples from light by covering the humidity chamber with aluminium foil.
SECTION: Section 5: Measurement of infectivity in Vero cells
62. Mount coverslips on microscope slides with Prolong Diamond antifade (ThermoFisher Scientific) and image samples on a fluorescence microscope.

 

SECTION: Section 5: Measurement of infectivity in Vero cells
61. Add 500 μl PBS-T (0.1% Tween-20) to each coverslip and incubate for 5 minutes at RT. Aspirate PBS-T solution and repeat step 61 two more times. Protect samples from light by covering the humidity chamber with aluminium foil.
SECTION: Section 5: Measurement of infectivity in Vero cells
60. Aspirate dye staining solution.
SECTION: Section 6.2: Measuring genomic DNA content of E. intestinalis parasites: Quantitative PCR (qPCR) of genomic DNA
68. Prepare a qPCR reaction mixture to a total volume of 10 µL, consisting of 5 µL of 2X SYBR Green qPCR Master Mix , 3 µL of nuclease-free water, 1 µL of a specific primer set and 1 µL of the isolated genomic DNA sample. 

 

SECTION: Section 6.1: Measuring genomic DNA content of E. intestinalis parasites: Genomic DNA isolation
63. Resuspend 1 µL of Band 1, Band 2 and Purified spores in 100 µL of  Proteinase K-Zymolyase (PKZ) buffer. 

 

SECTION: Section 6.2: Measuring genomic DNA content of E. intestinalis parasites: Quantitative PCR (qPCR) of genomic DNA
69. Initiate the following qPCR: a denaturation step at 95 °C for 10 minutes, followed by 45 cycles of PCR amplification. Each cycle consists of denaturation at 95 °C for 15 seconds, annealing at 60 °C for 10 seconds, and extension at 72 °C for 30 seconds. 
SECTION: Section 6.2: Measuring genomic DNA content of E. intestinalis parasites: Quantitative PCR (qPCR) of genomic DNA
70. Post-amplification, a melting curve analysis is performed. The PCR product is first re-annealed by heating to 95 °C (at 4.8 °C/s, hold 15 s) then cooling to 60 °C (at 2.5 °C/s, hold 1 s). The melt curve analysis is performed by heating from 60 °C to 95 °C at a rate of 0.06 °C/s, with acquisitions performed at every 0.1 °C. Product identity was confirmed by Sanger sequencing initially, and subsequently by comparison of melting profiles.

 


 

SECTION: Section 6.1: Measuring genomic DNA content of E. intestinalis parasites: Genomic DNA isolation
67. Denature samples by boiling at 95 °C for 5 minutes. 
SECTION: Section 6.1: Measuring genomic DNA content of E. intestinalis parasites: Genomic DNA isolation
66. Add 2 µL of Zymolyase to the samples and incubate at 30 °C for 60 minutes. This will digest the spore wall. 
SECTION: Section 6.1: Measuring genomic DNA content of E. intestinalis parasites: Genomic DNA isolation
65. Denature samples by boiling at 95 °C for 5 minutes. 
SECTION: Section 6.1: Measuring genomic DNA content of E. intestinalis parasites: Genomic DNA isolation
64. Add 2 µL of Proteinase K and incubate at 50 °C for 60 minutes.