Sucrose

To induce MAFLD and NASH, mice were fed a western diet (WD) high in fat, sugar and cholesterol as described previously (Ganz et al., 2015 (link)). This consisted of 58% fat, 1% cholesterol (Research Diets; D09061703i) and drinking water was supplemented with 23.1g/L fructose (MPBio) and 18.9g/L sucrose (VWR). Control mice were fed a standard diet with 11kcal% fat with corn starch (D12328i; Research Diets).

Spreads were prepared as previously described [42 (link), 61 (link), 86 (link)]. Briefly, testes were taken off from juvenile euthanized mice and the tunica albuginea was removed. Next, the seminiferous tubules were placed in DMEM high glucose (Euroclone, ECM0101L) and disrupted and mixed using a razor blade. The supernatant was centrifuged at 7200 rpm for 1 min and the pellet was resuspended in 0.5 M sucrose (VWR, 27480.294). Cell suspension was fixed on slides (Thermo Sientific, Menzel-Glaser Superfrost Plus, J1800AMNZ) using 1% paraformaldehyde (PFA) (ChemCruz, SC-281692)/0.015% Triton X-100 (Sigma-Aldrich, 9002-93-1)/dH2O pH 9.2) and incubated for 2 h in a humidified chamber at room temperature (RT). When the slides were completely fixed, they were washed twice with Washing Buffer 2 [WB2, 0.4% Photo-Flo (Kodak Professional 200 Solution, 1464510)/dH2O] and left air-drying at RT. Slides were either stained soon after or stored at − 80 °C for up to 6 months.

Three molecules were added to L. bulgaricus at the same molar concentration (0.58 M): glycerol 5.2% w/v (Sigma-Aldrich, France), sucrose 20% w/v (VWR Chemicals, France) and dimethyl sulfoxide, DMSO 4.5% w/v (VWR Chemicals, France), corresponding to molar concentrations currently employed for the production of starter cultures. Each aqueous solution was prepared in saline water (NaCl 0.15 M) with 0.2% of Snowmax (in order to facilitate ice nucleation) and sterilized at 121°C for 15 min.

The applied protective media consisted of 0.1 M phosphate buffer (PB) containing the protective agents sucrose (VWR International AG, Switzerland), inulin (RPN Foodtechnology AG, Switzerland), and glycerol (VWR International AG). Prior to preparation, components of PB and the used protectants were stored in an anaerobic chamber (10% CO₂, 5% H₂, 85% N₂) (Coy Laboratories, USA) overnight to remove residual oxygen. To prepare PB, sodium dihydrogen phosphate (6.0 g liter⁻¹) and sodium hydrogen phosphate (7.1 g liter⁻¹) (both from Sigma-Aldrich Chemie GmbH, Switzerland) were dissolved in oxygen-free distilled water. The pH was adjusted to 6.8 after the addition of the reducing agents cysteine-HCl and riboflavin (Sigma-Aldrich Chemie GmbH) at final concentrations of 1 g liter⁻¹ and 0.3 g liter⁻¹, respectively, to counter potential oxygen exposure during processing and storage (28 (link), 70 (link)). All three protectants (glycerol [15% {vol/wt}] and sucrose and inulin [both at 5% {wt/vol}]) (GSI) were dissolved in PB for cryopreservation, whereas a protective medium containing only sucrose and inulin (both 5% [wt/vol]) (SI) was used in the lyophilization trials. Protective media were filter sterilized, covered in aluminum foil for protection from light, and stored in an anaerobic chamber before use.

Agarose (Fischer Scientific, BP1356–100), Bovine Serum Albumin (Sigma, A4503), Fibronectin (Sigma, F1141), dextran fluorescein (Invitrogen, D1820), dextran tetramethylrhodamine (Molecular Probes life technologies, D3312), Gelatin (Sigma, G1890), Methylcellulose (Sigma, M0387–100), Mowiol 40–88 (Fluka, 81386), Penicillin-Streptomycin (Sigma, P4458), Sucrose (VWR, 27480.294). Danilchick’s medium 1 × (DFA): NaCl (53 mM), NA2CO3 (5 mM), K Gluconate (4.5 mM), Na Gluconate (32 mM), MgSO4–7H20 (1 mM), CaCl2 (1 mM), BSA 0.1%. MEMFA: MOPS (1 mM), EGTA (2 mM), MgSO4 (1 mM), Formaldehyde (3.7%). Normal Amphibian Medium (NAM): NaCl (110 mM), KCl, (2 mM), Ca(CO3)2 (1 mM), MgSO4 (1 mM), EDTA (0.1 mM), NaHCO3 (1 mM), Sodium Phosphate (2 mM).NTMT: NaCl (0.1 M), TrisHCl pH9.5 (0.1 M), MgCl2 (50 mM), Tween 0.1%. Phosphate Buffer Saline 1 × : NaCl (137 mM), KCl (2.7 mM), Na2HPO4 (10 mM), KH2PO4 (1.8 mM), CaCl2–2H2O (1 mM), MgCl2–6H2O (0.5 mM). Human Sdf1 (Calbiochem, 572300; used at 0.5 μg/mL in solution). Mouse Semaphorin-3A-Fc (R&D Systems, 5926-S3; used at 15, 30 or 60 ng/mL coated or added in solution), mouse Semaphorin-3F-Fc (R&D Systems, 3237-S3; used at 120, 240 or 480 ng/mL coated).

The EDTA-whole blood samples were thawed at room temperature and 100 µL were collected and mixed with Schneider’s insect medium (Sigma-Aldrich, Inc., St. Louis, MO, USA) supplemented with 10% of bovine calf serum (Biological Industries, Kibbutz Beit Haemek, Israel), 5% of sucrose (VWR International, Ltd., Poole, UK) and amphotericin B (Biological Industries, Kibbutz Beit Haemek, Israel) to a final concentration of 2.5 µg/mL [63 (link),64 (link)]. For liquid cultures, 200 μL of blood sample was added to 2 mL of medium and for solid cultures, 200 μL of the homogenized solution was diluted 1:1 in a liquid medium and seeded onto chocolate agar plates (Novamed, Ltd., Jerusalem, Israel), and incubated at 37 °C in a 5% CO₂ atmosphere. The plates were screened for the growth of suggestive Bartonella-like colonies (i.e., slow-growing, creamy or dry rounded colonies) after 3 days post-incubation, and then every 48 h, for 42 days [65 (link)].

Bacterial strains and plasmids used in this study are shown in Tables S3 and S4 in the supplemental material, respectively. Shigella spp. and E. coli were grown in tryptic soy broth (TSB; Sigma) and lysogeny broth (LB; Invitrogen), respectively, and 1.5% (wt/vol) agar (Oxoid) was added for solid media. Antibiotics were used at the following concentrations: carbenicillin, 100 μg/mL; chloramphenicol, 5 μg/mL (for S. flexneri) or 20 μg/mL (for S. sonnei); kanamycin, 50 μg/mL; streptomycin, 100 μg/mL. CR was added to TSB agar at a final concentration of 0.01% wt/vol (CR⁻ TSA). For sucrose selection, 1% (wt/vol) bacto-tryptone (Sigma), 0.5% (wt/vol), yeast extract (Sigma), and agar were autoclaved in water, and sucrose (VWR, a final concentration of 10%, wt/vol) added before pouring plates.