D. discoideum strains and plasmids are summarized in Supplementary Table 1. D. discoideum Ax2(Ka) was 407
axenically cultured at 22°C in Hl5c medium (Formedium) supplemented with 100 U mL−1 of penicillin and 408
100 µg mL−1 of streptomycin (Invitrogen). D. discoideum JH10 was cultured similarly as Ax2(Ka), with the 409
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addition of 100 g/mL of Thymidine (Sigma). Cells were transformed by electroporation and selected with 410
the relevant antibiotic. Hygromycin, G418 and blasticidin were used at a concentration of 50, 5 and 5 µg 411
mL−1, respectively. D. discoideum Ax2(Ka) tsg101- and atg1- tsg101- were obtained by transformation with 412
the pSP72 KO vector kindly provided by Dr. L. Aubry56. JH10 alxA- and JH10 alg2a-/b- mutants were 413
kindly provided by Dr. L. Aubrey57, 58. Ax2(Ka) cells expressing GFP-Atg8 and AmtA-mCherry were 414
described in20 and47, respectively. Ax2(Ka) cells expressing GFP-Vps32, Vps4-GFP and GFP-Tsg101 were 415
generated by transformation with the vectors described in Supplementary Table 1. Cells expressing GFP-416
Plin were obtained upon transformation with the pDNeoGFP-Plin construct described in59. 417
418
Mycobacteria strains, culture and plasmids 419
M. marinum strains and plasmids are summarized in Supplementary Table 1. M. marinum (M strain) wt and 420
ΔRD1 were kindly provided by Dr. L. Ramakhrisnan20. Mycobacteria were cultured in 7H9 (Difco) 421
supplemented with glycerol 0.2% (v/v) (Biosciences), Tween-20 0.05% (v/v) (Sigma) and OADC 10% (v/v) 422
(Middlebrock). mCherry-expressing bacteria were obtained by transformation with pCherry10, which was 423
kindly obtained from Dr. L. Ramakrishnan60, and cultured in the presence of 50 µg/mL-1 hygromycin 424
(Labforce). Luminiscent bacteria harbored the pMV306::lux plasmid48 and were cultured in presence of 50 425
µg mL-1 kanamycin (AppliChem).
426 427
Infection assay 428
Infections were performed as previously described61. In brief, M. marinum bacteria were washed in Hl5c 429
and centrifuged onto adherent D. discoideum cells. After additional 20-30 min of incubation, extracellular 430
bacteria were washed off and the infected cells resuspended in Hl5c containing 5 U mL-1 of penicillin and 431
5 µg mL-1 of streptomycin (Invitrogen). For live microscopy, mCherry expressing or unlabeled bacteria 432
were used. To monitor bacteria intracellular growth in D. discoideum, luciferase-expressing M. marinum20, 433
49 were used.
434 435
18 Intracellular growth measurement
436
Growth of luminescent bacteria was measured as described previously20, 49. Briefly, dilutions of 0.5-2.0 × 437
105 D. discoideum cells infected with M. marinum pMV306::lux were plated on a non-treated, white F96 438
MicroWell plate (Nunc) and covered with a gas permeable moisture barrier seal (Bioconcept).
439
Luminescence was measured for 72 h at 1 h intervals with a Synergy Mx Monochromator-Based Multi-440
Mode Microplate Reader (Biotek). The temperature was kept constant at 25°C.
441 442
Live fluorescence microscopy 443
Infected or uninfected D. discoideum were plated in 35 mm Glass Bottom Dishes (MatTek) or in 4-wells µ-444
slides (Ibidi). To induce damage in D. discoideum membranes, cells were incubated with 2.5-5 µM of 445
digitonin (Sigma), 5 mM of LLOMe (Bachem), 200 µM of GPN or 60 mg mL-1 of purified recombinant 446
ESAT-6 (BEI Resources). To visualize the lumen of endocytic compartments, 0.5 mg mL-1 of 70 KDa 447
TRITC-Dextran or 10-15 µg mL-1 of 10 kDa Alexa Fluor 647 Dextran (Molecular Probes) were added at 448
least 3 h prior to visualization of the sample. To detect neutralization of endocytic vesicles, 0.2 mM of 524 449
Da HPTS (Molecular Probes) was added at least 3 h prior to the visualization of the sample. To detect 450
plasma membrane damage, 5 µM of Annexin V Alexa Fluor 594 conjugate (Molecular Probles) was added 451
to cells in the presence of 2.5 mM Ca2+. To chelate Ca2+, 5 mM of EGTA (Fluka) or 5 mM of EGTA and 452
250 µM of BAPTA-AM (Sigma) were added at least 20 min before imaging. Bacteria not expressing a 453
fluorescent reporter were stained with 12.5 µM of Vybrant DyeCycle Ruby stain (Molecular Probes) directly 454
on the µ-dish prior to microscopic inspection. Live microscopy was performed with a Spinning Disc 455
Confocal Microscope [Intelligent Imaging Innovations Marianas SDC mounted on an inverted microscope 456
(Leica DMIRE2)] with a glycerin 63x or an oil 100x objective. Generally, z-stacks from 3 to 10 slices of 1 457
µm or 1.5 µm were acquired. For time-lapse experiments, images were acquired from every 15 s to several 458
min. Image analysis was performed using ImageJ.
459 460 461
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Cell fixation and immunofluorescence stainings 462
Samples were fixed in ultracold methanol as already described62. Briefly, D. discoideum cells on coverslips 463
were quickly fixed by immersion in -85°C methanol using a Freezing Dewar (FH Cryotec, 464
Instrumentenbedarf Kryoelektronenmikroskopie). Subsequent immunofluorescence staining was performed 465
as described62. Rabbit anti-Atg8 was previously described20, anti-p8063 was purchased from the Geneva 466
Antibody Facility (http://www.unige.ch/antibodies), the anti-Ub (FK2) monoclonal antibody was from Enzo 467
Life Sciences20. Nuclei were stained with DAPI (Molecular Probes). Cells were embedded using 468
ProlongGold antifade (Molecular Probes). Images were acquired with a LSM700 or LSM780 microscope 469
(Zeiss) using an oil 63x objective. Image analysis was performed using ImageJ.
470 471
Transmission Electron Microscopy (TEM) 472
Sample preparation for TEM was performed as described in64. Briefly, D. discoideum cells were fixed in a 473
6 cm dish in 2% (w/v) glutaraldehyde in Hl5c for 1 h and stained with 2% (w/v) OsO4 in imidazole buffer 474
0.1 M for 30 min. Cells were detached with a cell scraper and washed 3 times with PBS. Subsequent sample 475
preparation was performed at the Pôle Facultaire de Microscopie Ultrastructurale (University of Geneva).
476
Samples were incubated in 2% (v/v) of Milloning buffer and rinsed with distilled water. Then, they were 477
incubated in 0.25% (w/v) uranyl acetate overnight and rinsed with distilled water. Samples were dehydrated 478
using increasing concentrations of ethanol, then in propylene oxide for 10 min and finally embedded in 50%
479
Epon-propylene oxide for 1h, followed by incubation overnight in pure Epon. Samples were embedded in 480
2% agar for subsequent sectioning in an ultramicrotome and placed on TEM grids. Finally, sections were 481
visualized in a Tecnai 20 electron microscope (FEI Company, Eindhoven, The Netherlands).
482 483
Focus Ion Beam Scanning Electron Microscopy (FIB-SEM) 484
Initial sample preparation was performed similarly as for TEM and sent to the Pôle Facultaire de 485
Microscopie Ultrastructurale (University of Geneva). Subsequent contrast enhancement, dehydration and 486
resin embedding was performed as described in65 Samples were visualized in a Helios DualBeam NanoLab 487
20
660 SEM (Fei Company, Eindhoven, The Netherlands). 3 D reconstitutions were performed using the 488
LimeSeg plugin from ImageJ.
489 490
Statistics 491
All graphs were plotted and statistical analysis were performed using Prism. In all quantifications, the mean 492
and standard deviation is shown. Two-tailed t-test or 2-way ANOVA was used (n.s: non-significant, *: p-493
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629
Acknowledgements 630
We would like to acknowledge Dr. Laurence Aubry for her generous gift of antibodies and D. discoideum 631
ESCRT mutant strains, Dr. Sonia Arafah for the generation of the tsg101- mutant and preliminary work on 632
intracellular growth measurements, Ms Iryna Nikonenko and the PFMU platform of the Centre Medical 633
Universitaire for processing the samples for TEM and FIB-SEM, and the Bioimaging Platform of the 634
Faculty of Sciences. LG and MH were supported by a grant from the Deutsche Forschungsgemeinschaft 635
(HA3474/3-2), and JSK by a Royal Society University Research Fellowship (UF140624). The TS lab is 636
26
supported by grants from the Swiss National Science Foundation (310030_149390 and 310030_169386) 637
and the SystemsX.ch initiative grant HostPathX.
638
639
Authors contributions 640
Conceived and designed the experiments: ATLJ, TS. Performed the experiments: ATLJ, FL, ECM.
641
Analysed the data: ATLJ, ECM. Contributed reagents/material/analysis tools: ATL, FL, LG, MH, JSK.
642
Wrote the original draft: ATLJ, TS. Reviewing & editing: ATLJ, ECM, MH, JSK, TS.
643 644
Competing interests 645
The authors declare no competing interests 646
. CC-BY-NC-ND 4.0 International license peer-reviewed) is the author/funder. It is made available under a
The copyright holder for this preprint (which was not .
http://dx.doi.org/10.1101/334755 doi:
bioRxiv preprint first posted online May. 30, 2018;