Bacterial Toxin Research Citations

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

… The column was washed with T205N100P0.1 followed by T205N100P0.1 plus 20 mM imidazole and eluted with T205N100P0.1 with 150 mM imidazole. Peak fractions were pooled and diluted tenfold with T20P0.1, loaded onto a Source Q anion exchange column, and eluted by a 0-1M NaCl gradient. Purified EF was concentrated to approximately 20 mg/mL and frozen at -80C. Protein quantitation was performed using extinction coefficients calculated from the known primary sequence of each protein on the Expasy ProtParam webserver. PA was purchased from List Labs. …

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

Materials and Methods

Anthrax lethal factor (Product #172 or #169), are products of List Biological Laboratories, Inc. The 96-well, black, flat bottom, non binding plates used for the fluorescent plate assay were from Corning (cat # 3991). Bovine plasma (cat # 7310806) was
purchased from Lampire Biological Laboratories.

Sample Preparation: Stock solutions of the fluorogenic substrates, MAPKKide® Plus (± biotin), were made 2.5 mM in
DMSO based on the peptide content determined by elemental analysis. The substrates were diluted in assay buffer: 20 mM
HEPES, pH 8.0 containing 0.1% Tween-20.

LF Activity Assays:
A rapid microplate assay method was evaluated for two ranges of LF: 10 to 1000 pg LF/ml 1:10 diluted bovine plasma and 5 to 250 ng/ml 1:5 diluted bovine plasma. Dilution of the bovine plasma was necessary in order to minimize background.
For the method detecting higher levels of LF (5 to 250 ng/ml 1:5 diluted bovine plasma), 10 μM MAPKKide Plus was added directly to the 1:5 diluted bovine plasma, and the assays were run at 37°C using the kinetic mode of the plate reader with
readings at 1 or 3 minute intervals. The samples were run in triplicate with 9 replicate blanks. The limit of detection was
calculated from the normal distribution of the blank samples (mean + 3 stdev; n = 9).
For the range 10 and 1000 pg/ml 1:10 diluted plasma, 1.25 μM MAPKKide Plus was added directly to the diluted bovine
plasma and the time-dependent increase in fluorescence was monitored at 37°C hourly for 5 hours. For the blank samples
containing no LF there were 3 sets of quadruplicates and the standard deviation was calculated from these three sets. At
each time point, the plate was read 5 times to increase the precision of the fluorescence readings. The standard curve was
analyzed using a linear regression fit forcing the intercept through the mean value of the blanks. The limit of detection was
calculated from the normal distribution of the blank samples (mean + 3 stdev; n = 3 sets of quadruplicates) and calculated as
pg LF/ml plasma using the standard curve. Subsequently, 6 data sets were evaluated, 2 data sets per day for 3 consecutive
days. The data is presented as the average of these 6 data sets, each with 4 replicate samples and 12 replicate blanks. At
each time point, the plate was read 3 times to increase the precision of the fluorescence readings.

• Product #169L – Anthrax Lethal Factor (LF-A), Recombinant from B. anthracis Native Sequence
• Prodcut #172 – Anthrax Lethal Factor (LF), Recombinant from B. anthracis
• Product #532 – MAPKKide® Plus (AMC) Specific Substrate for Anthrax Lethal Factor

Lep-vesicle-recycling endosome-exocyst complex inhibition tests:

The cell monolayers were treated with 35 nM edema factor or lethal factor plus 70 nM protective antigen (EF + PA or LF + PA) of anthrax toxin (List Biological Laboratories, USA), the inhibitor (EF + PA) of Rab11 and inhibitor (LF + PA) of Sec15 to block Rab11-Sec15 binding, for 24 hr at 37C (Guichard et al., 2010), and then infected with L. interrogans strain Lai at MOI100 for 8 hr. The subsequent steps and detection of Lep-vesicle-Rab11-Sec15/Sec3 co-localization were the same as above. …

• Product #171 – Anthrax Protective Antigen (PA), Recombinant from B. anthracis
• Product #178A – Anthrax Edema Factor (EF), Recombinant from B. anthracis
• Product #169 – Anthrax Lethal Factor (LF-A), Recombinant from B. anthracis Native Sequence