Bacterial Toxin Research Citations

Reagents

Lethal factor (LF) was purchased from List Biological Laboratories (#169, recombinant from B. anthracis)

• Product #169L – Anthrax Lethal Factor (LF-A), Recombinant from B. anthracis Native Sequence

Protein expression and purification

Upon addition of double molar excess of trypsin inhibitor (Sigma Aldrich), PA7 was further purified by size exclusion chromatography using a Superdex 200 column (GE Healthcare). Lyophilized LF (List Biological Lab. Inc., Lot#1692A1B) were resuspended in water according to the manufacturer’s manual and mixed with PA7 in a molar ration of 10:1. …

• Product #169L – Anthrax Lethal Factor (LF-A), Recombinant from B. anthracis Native Sequence

Reagents

… Lethal Toxin was from List Biological Laboratories. …

• Product #171E – Anthrax Protective Antigen (PA), Recombinant from B. anthracis

Author did not specify which Lethal Factor (LF) was utilized; List Labs provides the following LF:

• Product #172 – Anthrax Lethal Factor (LF), Recombinant from B. anthracis
• Product #169L – Anthrax Lethal Factor (LF-A), Recombinant from B. anthracis Native Sequence

Chemicals

… PA63 protein was prepared from PA83 using furin (53) or purchased from List Biological Laboratories (Campbell, CA) in the purified form. …

• Product #174 – Anthrax Protective Antigen, Activated (PA 63) from B. anthracis

… 1:1,000 for antiEF and antiLF sera, and antiPA antibody (List Labs, Campbell, California). …

Chemicals and Reagents:

… Recombinant LF was from List Biological Laboratories (Campbell, California). …

Author did not specify which Lethal Factor (LF) was utilized; List Labs provides the following LF:

• Product #172 – Anthrax Lethal Factor (LF), Recombinant from B. anthracis
• Product #169L – Anthrax Lethal Factor (LF-A), Recombinant from B. anthracis Native Sequence

… For intoxication of cells, media was replaced over 105 cells previously seeded in a 12-well plate. 31.7 nM PA alone (List Labs, #171E) or …

• Product #171E – Anthrax Protective Antigen (PA), Recombinant from B. anthracis

Reagents and antibodies

… For cell culture experiments, Val-boroPro was resuspended in DMSO containing 0.1% TFA to prevent compound cyclization. Anthrax Lethal Factor (Catalog #172B) and PA (Catalog #171E) were purchased from List Labs, …

LDH cytotoxicity assays

For LDH release experiments involving primary macrophages, mBMDM and rBMDM were isolated from the femurs and tibias of 79-week-old male and female mice and 712-week-old male rats. 110⁷ cells were plated on 10cm Petri dishes. mBMDMs were differentiated in DMEM with 10% FBS and 20% L Cell medium for 6 days and rBMDMs in 30% L Cell medium for 79 days. Macrophages were scraped and reseeded at 0.2510⁶ cells/well in 12-well plates in Opti-MEM and incubated overnight before treatment. Cells were then treated as indicated with compounds or LT. …

In vitro and in cell LF cleavage assays

HEK 293T cells were seeded at 0.510⁶ cells/well in six-well plates and transiently transfected with 2g of the indicated plasmids using FuGENE HD. For the in vitro assay, lysates were harvested after 48h and normalized to 1mg/mL using the DC Protein Assay kit (Bio-Rad). These lysates were then incubated with LF (1g/mL) for the indicated time periods at 25C quenched by boiling with 2SDSPAGE sample buffer at the indicated times, separated by SDSPAGE, immunoblotted, and visualized using the Odyssey Imaging System (Li-Cor). For the in cell assay, after 48h after transfection cells were treated with LT (1g/mL, 6h) before lysates were harvested for immunoblotting as described above.

• Product #171E – Anthrax Protective Antigen (PA), Recombinant from B. anthracis
• Product #172 – Anthrax Lethal Factor (LF), Recombinant from B. anthracis

Stimulation conditions:

iMos were primed with 200ng/mL LPS (Ultrapure LPS, Invivogen) for 3h. Inflammasome activation was performed with 10M nigericin (Life Technologies) and 5mM ATP (Sigma) for 1h or with 1mg/mL Silica (US-Silica), 1mM LeuLeuoMe (ChemImpex), lethal toxin (1g/mL lethal factor (List Biologicals), 1g/mL, protective antigen (List Biologicals), poly(dA:dT) (20ng/mL+0.5% lipofectamin 2000), LFn-PrgI (4g/mL (M. Geyer)+protective antigen 1g/mL) for 3 to 4h, 12 or 25g/mL PGN from S. Aureus (Invivogen) for 20h, 20g/mL R837 (Invivogen) for 1.5h, cytosolic LPS (Cholera toxin subunit B (CTB) (20g/mL+2g/mL LPS), or with DOTAP (3.75g/mL+750ng/mL LPS)) for 16h. …

• Product #171E – Anthrax Protective Antigen (PA), Recombinant from B. anthracis

Author did not specify which Lethal Factor (LF) was utilized; List Labs provides the following LF:

• Product #172 – Anthrax Lethal Factor (LF), Recombinant from B. anthracis
• Product #169L – Anthrax Lethal Factor (LF-A), Recombinant from B. anthracis Native Sequence

Toxins:

…  Recombinant EF and PA from Bacillus anthracis were purchased from List Biological Laboratories, Inc., Campbell, CA. …

Signal Transduction Experiments:

Zymosan particles were opsonized by incubating the particles with 50% human serum for 30 min at 37 C. The particles were then washed and dissolved in DMEM with FCS before use for treatment of cells (100 g of zymosan particles per 10⁶ cells). To investigate the effects of toxin-provoked cAMP intoxication on opsonophagocytic signaling, 3 10⁶ THP-1 cells were preincubated with CyaA (150 ng/ml; 0.85 nM) for 30 min or ET (625 ng/ml; 7.04 nM EF + 2500 ng/ml; 30.24 nM PA) for 6 h, respectively (based on cAMP ELISA experiments to have similar cAMP intoxication levels, cf. Figure 1A,B). The cells were then treated with 300 g of serum opsonized or unopsonized zymosan as positive and negative controls for 30 min, respectively.

• Product #171E – Anthrax Protective Antigen (PA), Recombinant from B. anthracis
• Product #178A – Anthrax Edema Factor (EF), Recombinant from B. anthracis