Bead ruptor 12

C. jejuni cells were disrupted by bead beating (Bead Ruptor 12, Omni International) at maximal intensity for 30 s and the crude cell extracts were used for SDS/PAGE. For Western blot analysis, proteins were electroblotted on to nitrocellulose membranes (Amersham Protran 0.45 μm NC, GE Healthcare) for 35 min at 15 V using the Transblot SD semi-dry transfer apparatus (Bio-Rad Laboratories). His-tagged protein was detected with His-tag monoclonal antibody (#70796-3, Novagen) and Goat Anti-Mouse IgG horseradish peroxidase (HRP) Conjugate (#71045-3, Novagen) via the associated peroxidase activity using either chloronaphthol or the SignalFire ECL Reagent kit (Cell Signaling Technology).

Total RNA from DNA/RNA Shield Blood Collection Tubes was isolated by using Quick-DNA/RNA Blood Tube Kit with DNase treatment according to manufacturer’s provided protocol (Zymo Research). Total RNA was isolated by using TRIzol Reagent and the Direct-zol RNA MiniPrep Kit with DNase treatment according to manufacturer’s provided protocol (Zymo Research). Mouse tissues were homogenized in TRIzol (Ambion) with 1.5 mm zirconium beads in a Bead Ruptor 12 (OMNI International). Total RNA was isolated from thymus using PolyTron (Kinematica) homogenization in TRI Reagent (Sigma) followed by treatment with the RNeasy Kit (Qiagen) according to the manufacturer’s instructions. RNA was quantitated on a Nanodrop, Quant-iT RiboGreen RNA Assay Kit (Thermo Fisher) and/or Qubit RNA BR Assay Kit (Thermo Fisher Scientific). RNA quality was assessed using a Bioanalyzer 2100 (Agilent Technologies).

Litter and sex matched C57BL/6J mice bred from Mcoln2^+/− parents by the Duke DLAR breeding core were infected when 10–18 weeks old with S. Typhimurium 14028s sub-cultured 1:33, grown for 160 min to late-log (OD₆₀₀ 1.7–1.9), and then washed twice with sterile PBS. Bacteria were re-suspended in PBS at 10,000/mL based on OD₆₀₀ and mice were infected via intraperitoneal injection with 100 μL of PBS containing 1,000 CFUs. For competitive infections, the initial 1:1 ratio used 500 CFUs of each Amp^R wild-type (+pWSK29; DCK483) and Kan^R mutant (ΔmgtAΔmgtB + pWSK129; DCK1132) S. Typhimurum. All inoculums were verified by plating for CFUs. All mice were monitored twice daily for morbidity. Spleens were harvested 4 dpi, homogenized by bead beating with ZrO beads (GlenMills #7305-000031) with a Bead Ruptor 12 (Omni #19-050A), and a serial dilution was plated for CFUs on LB + Amp (100 μg/mL) or Kan (50 μg/mL). Competitive index was calculated as ratio of Kan/Amp CFUs.

SCFA were measured in the feces of all the animals at week 11 for the 12-week study and at week 2 for the 2-week studies. SCFA were extracted according to a protocol previously published by García Villalba et al.^{60 (link)} with some modifications. Right after collection, feces were weighed and 1 mL of 0.5% phosphoric acid was added per 100 mg of material. Fecal suspensions were homogenized 2 min with a Bead Ruptor 12 (Omni International, Kennesaw, GA, USA), then centrifuged at 18,000 × g for 10 min at 4 °C. The supernatant was collected and an equal volume of ethyl acetate, spiked with internal standard 4-methylvaleric acid were added. To extract SCFA, samples were mixed 2 min at 2400 rpm using a VWR VX‑2500 Multitube Vortexer (VWR, Radnor, PA, USA), then centrifuged at 18,000 × g for 5 min at 4 °C. The organic phase was transferred to an autosampler vial for for gas chromatography analysis. A 5-point calibration curve were prepared with a mix of acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid, and internal standard 4-methyl valeric acid. SCFA quantification was performed on a Shimadzu GC 2010 Plus equipped with a Nukol Supelco capillary GC column (30 m × 0.25 mm id, 0.25 µm) and a FID detector.

C. neoformans cultures were harvested and washed three times in 1X PBS and resuspending in 3 ml/g cell pellet of lysis buffer (50 mM Hepes [pH 7.5], 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, and 1X yeast-specific protease inhibitor [Sigma-Aldrich]). One milliliter resuspended cells were added to 2 ml screw cap Eppendorf tubes with added acid washed, autoclaved glass beads (425–600 μm, Sigma-Aldrich), and subjected to cell lysis by a Bead Ruptor 12 (Omni International) for 30 s on, 2 min off, ten cycles at 4 °C with chilling on ice in between cycles. Cell lysates were clarified by centrifugation at 5000g for 5 min, followed by centrifugation at 12,000g for 20 min. Clarified supernatants were assessed for protein content by BCA assay (Thermo Scientific) and proteins separated by 4 to 20% SDS-PAGE. Proteins were detected by immunoblotting using standard methods as previously described with the indicated antibodies.

Flushed lungs were obtained from storage at −80°C and placed on ice. The reagents, Pierce's Tissue Protein Extraction Reagent, Sigma's Phosphatase Inhibitor Cocktail 2, Sigma's Phosphatase Inhibitor Cocktail 3, and Sigma's Protease Inhibitor Cocktail were prepared per manufacturer's instructions. Tissue samples (25–30 mg) were separated and placed in the Bead Ruptor₁₂ (Omni International), run at High Speed for 45 s, and then transferred to ice ×3 cycles. Homogenized samples were incubated for 30 min on ice, then centrifuged at 10,000 g for 5 min. 5‐HT levels were measured using the 5‐HT ELISA kit (ALPCO) following the manufacturer's instructions.