Bbx24b

O. tsutsugamushi strain UT-76 (a Karp-like strain from Thailand) was propagated in T25 cell culture flasks containing a confluent monolayer of L929 cells which were grown in DMEM supplemented with 10% FBS at 35°C with 5% CO₂. After 7 days of infection the cells were sub-cultured onto fresh L-929 cell monolayers. The infected flasks were harvested by scraping using a sterile inoculating loop in 1ml spent medium and disrupted to release intracellular bacteria by lysing in a bullet blender (BBX24B, Bullet Blender Blue, Nextadvance, USA) used at power 8 for 1 min. The lysed bacteria were added to a T25 flask (100 μl/flask) containing an uninfected L-929 monolayer at 70–100% confluence. Infected flasks were passaged at a ratio of 1:5 (original culture flask: subculture flask). Bacteria were sub-cultured for a total of <20 passages, where passage 0 was the original clinical isolate.
Antibiotics were only used in the experiment testing the effect of antibiotic addition. Here antibiotics were used at the following concentrations: chloramphenicol 150 μg/ml, penicillin G 100 μg/ml, penicillin G + streptomycin (premix from Sigma-Aldrich) 125 μg/ml + 200 μg/ml.
Cell lines and bacteria were tested for the presence of mycoplasma and confirmed to be mycoplasma-free using the VenorGeM PCR detection kit (Minerva biolabs).

The supernatant were removed from infected flasks and replaced with 6–8 ml pre-warmed media. Infected cells were harvested by mechanical scraping and then lysed using a bullet blender (BBX24B, Bullet Blender Blue, Nextadvance, USA) operated at power 8 for 1 min. Host cell debris was removed by centrifugation at 300xg for 3 minutes, and the supernatant was filtered through a 2.0 μm filter unit (Puradisc, GE Whatman, USA). 10 μl of 1.4 μg/μl DNase (Deoxyribonuclease I from bovine pancreas, Merck, UK) was added per 1 ml of bacterial solution, then incubated at room temperature for 30 minutes. This procedure removed excess host cell DNA. The bacterial sample was then isolated by centrifugation at 14,000xg for 10 min at 4 ^oC, and washed two times with 0.3M sucrose (Merck). After the washing steps were completed DNA was extracted using a QIAGEN DNeasy Blood & Tissue Kit (QIAGEN, UK) following the manufacturer’s instructions.
Purified DNA samples were analysed by gel electrophoresis using 0.8% agarose gel, in order to assess the DNA integrity. The yield of genomic DNA was quantified using a nanodrop (Nanodrop 2000, Thermo Scientific, UK) and Qubit Fluorometric Quantitation (Qubit 3.0 Fluorometer, Thermo Scientific).

For Western blot procedures, cardiac tissues were homogenized with RIPA buffer (Thermo Fisher Scientific, RIPA buffer 89900) and protease inhibitors (Roche, 11-836-170-001) using a bullet blender (Next Advance, BBX24B) at 4°C for 10 minutes. Protein concentrations of supernatants were collected after centrifugation (20,000g, 4°C, 20 minutes) and quantified using Pierce BCA Protein Assay Kits and Reagents (catalog 23225). Protein lysates in Lammeli sample buffer were loaded to 10 % SDS-PAGE, transferred to PVDF membrane, and then incubated at room temperature in Ponceau S (MilliporeSigma, P7170) for 10 minutes. Membranes were washed three times in distilled H₂O and then scanned to determine protein loading. Membranes were then blocked with 5% BSA in TSBT for 1 hour at room temperature. Specific proteins were detected by specific antibodies listed above and corresponding secondary antibodies. Signals were visualized by HRP-derived chemiluminescence (Amersham ECL Western Blotting Detection System, RPN2106) and film. Protein levels were quantified by ImageJ (NIH).