Contribution of the <i>csgA</i> and <i>bcsA</i> genes to <i>Salmonella enterica</i> serovar Pullorum biofilm formation and virulence

<p><i>Salmonella</i> biofilm formation is important to environmental stress resistance and virulence. However, the roles of the <i>csgA</i> and <i>bcsA</i> genes, which affect curli protein and cellulose production, respectively, in <i>Salmonella enterica</i> serovar Pullorum, are unknown. Here we constructed deletions in the <i>csgA</i> and <i>bcsA</i> genes in <i>S. enterica</i> serovar Pullorum strain S6702 and evaluated several aspects of biofilm formation and virulence. Δ<i>csgA</i> showed decreased production of curli fimbriae, while Δ<i>bcsA</i> had reduced cellulose production. Both mutants had a reduced ability to form biofilms. Δ<i>csgA</i> was reduced in adhesion and invasion to HeLa cells and exhibited decreased intracellular proliferation in HD11 macrophages. Δ<i>bcsA</i> exhibited increased proliferation in HD11 cells and replicated better in chicken spleens, as compared to the wild-type strain. Δ<i>csgA</i> virulence was attenuated in assays involving oral challenge of one-day-old chickens.</p

CaM is essential for Cd induction of ROS in neuronal cells.

<p>Indicated cells pretreated with/without TFP at indicated concentrations for 30 min (A), or infected with lentiviral shRNAs to CaM and GFP, respectively (B), were exposed to 0–20 µM Cd for 24 h, followed by ROS detection, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019052#s2" target="_blank">Materials and Methods</a>. Results are presented as mean ± SE; <i>n</i> = 6. ^a<i>P</i><0.01, difference vs. control group; ^b<i>P</i><0.01, difference vs. 10 µM Cd group; ^c<i>P</i><0.01, difference vs. 20 µM Cd group; ^d<i>P</i><0.01, CaM shRNA group vs. GFP shRNA group.</p

Inhibition of CaM by TFP attenuates Cd activation of MAPK/mTOR and apoptosis in neuronal cells.

<p>Indicated cells were pretreated with/without TFP at indicated concentrations for 30 min, and then exposed to Cd (10 and 20 µM) for 4 h (A, B) or 24 h (C). (A and B) Indicated cell lysates were subjected to Western blotting using indicated antibodies. The blots were probed for β-tubulin as a loading control. Similar results were observed in at least three independent experiments. (C) Cell viability for indicated cells was evaluated using one solution assay. Results are presented as mean ± SE; <i>n</i> = 6. ^a<i>P</i><0.05, ^b<i>P</i><0.01, difference vs. control group; ^c<i>P</i><0.01, difference vs. 10 µM Cd group; ^d<i>P</i><0.01, difference vs. 20 µM Cd group.</p

Cd-elevated [Ca^2<b>+</b>]_i induces ROS, triggering apoptosis of neuronal cells.

<p>PC12 cells, pretreated with/without BAPTA/AM, EGTA, and TFP at indicated concentrations for 30 min, were treated with/without 20 µM Cd for 24 h, followed by Western blot analysis using indicated antibodies. The blots were probed for β-tubulin as a loading control. Similar results were observed in at least three independent experiments.</p

Cd-elevated [Ca^2<b>+</b>]_i induces ROS in neuronal cells.

<p>Indicated cells were exposed to 0–20 µM Cd for 24 h after pretreatment with/without indicated concentrations of BAPTA/AM (A) or EGTA (B) for 30 min, followed by ROS detection, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019052#s2" target="_blank">Materials and Methods</a>. Results are presented as mean ± SE; <i>n</i> = 6. ^a<i>P</i><0.05, ^b<i>P</i><0.01, difference vs. control group; ^c<i>P</i><0.01, difference vs. 10 µM Cd group; ^d<i>P</i><0.01, difference vs. 20 µM Cd group.</p

Cd-induced neuronal apoptosis is associated with induction of [Ca^2<b>+</b>]_i elevation.

<p>(A and B) PC12 cells were treated with 0–20 µM Cd for 24 h, or with 0, 10 and 20 µM Cd for 0–24 h, and then loaded with 5 µM Fluo-3/AM for 30 min at 37°C in the dark, followed by measurement of [Ca²⁺]_i fluorescence intensity, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019052#s2" target="_blank">Materials and Methods</a>. (C and D) SH-SY5Y cells were exposed to 0–20 µM Cd for 24 h, or 10 µM Cd for 0–24 h, and then loaded with 2.5 µM Fluo-4/AM for 60 min at 37°C in the dark, followed by recording of the images under a fluorescence microscope. (E and F) Cell viability of PC12 cells, treated with 0–20 µM Cd for 24 h or with 20 µM Cd for 0–24 h, was evaluated by one solution assay. Results are presented as mean ± SE; <i>n</i> = 6. ^**<i>P</i><0.01 difference vs. control group.</p

Cd activates MAPK/mTOR pathways and neuronal apoptosis via induction of [Ca^2<b>+</b>]_i elevation.

<p>Indicated cells were pretreated with/without BAPTA/AM at indicated concentrations for 30 min, and then exposed to Cd (10 and/or 20 µM) for 24 h (A–C) or 4 h (D, E). (A) [Ca²⁺]_i in PC12 cells was determined by measuring Fluo-3/AM-labeled fluorescent intensity, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019052#s2" target="_blank">Materials and Methods</a>. (B) [Ca²⁺]_i was stained with Fluo-4/AM and visualized by fluorescence microscopy in SH-SY5Y cells. (C) Cell viability for PC12, SH-SY5Y, and primary neurons was evaluated using one solution assay. (D and E) Indicated cell lysates were subjected to Western blotting using indicated antibodies. The blots were probed for β-tubulin as a loading control. Similar results were observed in at least three independent experiments. Results (A and C) are presented as mean ± SE; <i>n</i> = 6. ^b<i>P</i><0.01, difference vs. control group; ^c<i>P</i><0.01, difference vs. 10 µM Cd group; ^d<i>P</i><0.01, difference vs. 20 µM Cd group.</p

Growth kinetics of the viruses in Vero, MDCK, CEF, and DEF cells.

<p>The cells were infected with the wild-type strain and the four rescue viruses at an MOI of 0.01 TCID₅₀/cell, and the culture media were harvested at the indicated times after infection. The virus titers at each time point are presented as the mean ± SD of duplicate experiments.</p

Primers for the mutagenesis of the NA and NS genes of the H5N1 AIV SY strain.

<p>Ba-NA-1^a, the restriction endonucleases site for BsaI is underlined.</p><p>Bm-NS-1^b<b>,</b> the restriction endonucleases site for BsmBI is underlined.</p

Real-time PCR primers for detection of the expression levels of immune-related genes in the PBMCs of mallard ducks.

<p>Real-time PCR primers for detection of the expression levels of immune-related genes in the PBMCs of mallard ducks.</p