Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Bacillus anthracis co-opts nitric oxide and host serum albumin for pathogenicity in hypoxic conditions

doi: 10.3389/fcimb.2013.00016

Figure Lengend Snippet: Supplementation of complete DMEM/F12 culture medium with BSA increases the toxicity of Sups of dSterne cultures grown in microaerobic conditions for 24 h. HSAECs were exposed to Sups for 2 h. Untreated cells served as control. OD 600 of Sups was measured in 96-well plate reader (200 μl per well). Error bars indicate 95% confidence intervals.

Article Snippet: Primary human small-airway lung epithelial cells (HSAECs) were from Cambrex, Inc., MD.

Techniques: Control

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Bacillus anthracis co-opts nitric oxide and host serum albumin for pathogenicity in hypoxic conditions

doi: 10.3389/fcimb.2013.00016

Figure Lengend Snippet: Antioxidant treatment of Sups protects HSAECs. The dSterne Sups grown in complete DMEM/F12 were incubated with 5 mM DTT for 30 min (A) or indicated concentrations of cysteine for 1 h (B) . HSAECs were exposed to the treated Sups for 2 h and the viability of cells was tested using Alamar Blue. In control experiments, addition of Cys had no effect on viability of untreated cells, and no cytoprotection was detected with other L - amino acids tested (Val, Leu, Ala) (not shown). Error bars indicate 95% confidence intervals.

Article Snippet: Primary human small-airway lung epithelial cells (HSAECs) were from Cambrex, Inc., MD.

Techniques: Incubation, Control

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Bacillus anthracis co-opts nitric oxide and host serum albumin for pathogenicity in hypoxic conditions

doi: 10.3389/fcimb.2013.00016

Figure Lengend Snippet: Inhibition of baNOS with L-NAME decreases toxicity of Sups to HSAECs. Static cultures of dSterne strain were grown in complete DMEM/F12 as described in Materials and Methods with or without the indicated concentrations of L-NAME. The table shows the ODs and pHs of 24-h cultures. The Sups were added for 2 h to HSAECs pretreated or not with the indicated concentrations of L-NAME for 1 h, and the viability of the cells was assayed with Alamar Blue. Controls included the culture medium with L-NAME without bacteria. All tested samples were titrated with HCl to the pH 5.1 of Sups without L-NAME. Error bars indicate 95% confidence intervals.

Article Snippet: Primary human small-airway lung epithelial cells (HSAECs) were from Cambrex, Inc., MD.

Techniques: Inhibition, Bacteria

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Bacillus anthracis co-opts nitric oxide and host serum albumin for pathogenicity in hypoxic conditions

doi: 10.3389/fcimb.2013.00016

Figure Lengend Snippet: Nitrosylated BSA (NO-BSA) is not acutely toxic. HSAECS were incubated with NO-BSA at indicated concentrations and pH for 2 h in DMEM/F12 medium, and the viability of HSAECs was tested using Alamar Blue. Error bars indicate 95% confidence intervals.

Article Snippet: Primary human small-airway lung epithelial cells (HSAECs) were from Cambrex, Inc., MD.

Techniques: Incubation

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Bacillus anthracis co-opts nitric oxide and host serum albumin for pathogenicity in hypoxic conditions

doi: 10.3389/fcimb.2013.00016

Figure Lengend Snippet: Blocking of free SH groups of BSA does not reduce the toxicity of dSterne Sup grown in the presence of BSA. The protein was dissolved in 10 ml of PBS, treated with 5 mM DTT for 30 min, and then dialyzed against two changes of 1 l of PBS for 24 h. The reduced BSA was then modified by NEM (125 μM) for 1 h at 37°C and dialyzed against two changes of 1 l of PBS overnight. The modified and mock-treated BSA was used to supplement the DMEM/F12 medium at 2 mg/ml. Bacterial cultures were grown for 24 h and Sups were tested for toxicity using HSAECs and the Alamar Blue viability assay. Error bars indicate 95% confidence intervals.

Article Snippet: Primary human small-airway lung epithelial cells (HSAECs) were from Cambrex, Inc., MD.

Techniques: Blocking Assay, Modification, Viability Assay

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Bacillus anthracis co-opts nitric oxide and host serum albumin for pathogenicity in hypoxic conditions

doi: 10.3389/fcimb.2013.00016

Figure Lengend Snippet: Permanganate treatment reduces the toxicity of dSterne Sup. The indicated concentrations of KMnO 4 were added for 1 h to the Sup grown in CSFM for 24 h, and toxicity of the treated Sup was tested after incubation with HSAECs for 2 h. Error bars indicate 95% confidence intervals.

Article Snippet: Primary human small-airway lung epithelial cells (HSAECs) were from Cambrex, Inc., MD.

Techniques: Incubation

Journal: PLoS ONE

Article Title: Bacillus anthracis Spore Entry into Epithelial Cells Is an Actin-Dependent Process Requiring c-Src and PI3K

doi: 10.1371/journal.pone.0011665

Figure Lengend Snippet: A – C , the effect of cytochalasin D on spore internalization by A549 ( A ), HeLa ( B ) and hSAECs ( C ). Cells were pretreated with different concentrations (0.01 – 10 µM for A549 cells, 1 and 10 µM for HeLa and hSAECs, respectively) of cytochalasin D (Cyto D) or the solvent control (Ctrl) for 1 hr and then performed fluorescent microscopic examination as described in . Relative internalization (%) is the ratio of intracellular spores vs. total adhered spores, normalized to the solvent control. The results are the mean ± SEM from at least two independent experiments. D – F , representative images of colocalization of F-actin with spores. A549 cells were incubated with Texas-Red labeled spores and then stained with phalloidin-Alexa Fluor 488. G , colocalization of F-actin with spores is inhibited by Cyto D. Cyto D (10 µM) was included in the colocalization examination as described in . The percentage of spores with enriched F-actin staining vs. total attached spores is denoted as %Colocalization. The results shown are combined from five independent experiments. ***, p< 0.0001, t test. Scale bars represent 5 µm. H – J , Cdc42 is the primary Rho-family GTPase required for spore uptake. A549 cells were transfected with pcDNA3.1(+) vector control (Ctrl), HA-T19NRhoA (T19NRhoA), HA-T17NRac1 (T17NRac1) or HA-T17NCdc42 (T17NCdc42). The expression of the transfected constructs was verified using western blot analysis 24 hr post transfection ( H ). Dominant negative GTPases were detected by anti-HA antibodies and secondary antibodies as described in . The actin level was used as a loading control. Spore uptake by ( I ) and adherence to ( J ) transfected cells were determined by gentamicin protection assays 24 hr post transfection as described in . Relative internalization (%) is the ratio of intracellular spores vs. total spores added, normalized to the solvent control. Relative association (%) is the ratio of associated spores (extracellular adhered + intracellular) vs. total spores added, normalized to the solvent control. The results shown are the mean ± SEM, combined from three independent experiments. **, p = 0.0018, t test.

Article Snippet: Clonetics® primary human small airway epithelial cells (hSAECs) (Cambrex) were maintained at 37°C in a humidified chamber with 5% CO 2 in SABM media supplemented with reagents from the SAGM SingleQuot kit (Cambrex), following instructions from the supplier.

Techniques: Solvent, Control, Incubation, Labeling, Staining, Transfection, Plasmid Preparation, Expressing, Construct, Western Blot, Dominant Negative Mutation