Primers used in this study.

<p>^a restriction sites are underlined</p><p>Primers used in this study.</p

Quantification of gene expression.

<p>Expression of genes involved in resistance and virulence were measured by qRT-PCR for each strain. Data were normalized to the housekeeping gene <i>dnaE</i>. Results are relative expression ratios compared to wild-type strain DE205B. *** <i>p</i> < 0.001; **<i>p</i> < 0.01; *<i>p</i> < 0.05. The error bars indicate standard deviations.</p

Bacterial strains and plasmids used in this study.

<p>Bacterial strains and plasmids used in this study.</p

IbeR Facilitates Stress-Resistance, Invasion and Pathogenicity of Avian Pathogenic <i>Escherichia coli</i>

<div><p>Systemic infections by avian pathogenic <i>Escherichia coli</i> (APEC) are economically devastating to poultry industries worldwide. IbeR, located on genomic island GimA, was shown to serve as an RpoS-like regulator in <i>rpoS</i> gene mutation neonatal meningitis <i>E</i>. <i>coli </i>(NMEC) RS218. However, the role of IbeR in pathogenicity of APEC carrying active RpoS has not yet been investigated. We showed that the APEC IbeR could elicit antibodies in infected ducks, suggesting that IbeR might be involved in APEC pathogenicity. To investigate the function of IbeR in APEC pathogenesis, mutant and complementation strains were constructed and characterized. Inactivation of <i>ibeR</i> led to attenuated virulence and reduced invasion capacity towards DF-1 cells, brains and cerebrospinal fluid (CSF) <i>in vitro</i> and <i>in vivo</i>. Bactericidal assays demonstrated that the mutant strain had impaired resistance to environmental stress and specific pathogen-free (SPF) chicken serum. These virulence-related phenotypes were restored by genetic complementation. Quantitative real-time reverse transcription PCR revealed that IbeR controlled expression of stress-resistance genes and virulence genes, which might led to the associated virulence phenotype.</p></div

Determination of bacterial virulence.

<p>Seven-day-old ducks were inoculated intratracheally with DE205B, ΔibeR, CΔibeR or ΔibeRibeA at 10⁷ colony-forming units (CFUs). Negative controls were injected with PBS. Survival was monitored until 7 days post infection.</p

IbeR was involved in invasion DF-1 cells by DE205B.

<p>Invasion assays were performed on DF-1 cells. Values are average of three independent experiments. The error bars indicate standard deviations. One-way ANOVA was performed for statistical significance analysis. *** <i>p</i> < 0.001; ** <i>p</i> < 0.01; * <i>p</i> < 0.05.</p

Expression of IbeR by Western blotting.

<p><b>(A)</b> Immunoblotting with anti-IbeR of total cell lysates from different APEC strains. Expression of IbeR was detected in wild-type strain DE205B and complementation strain CΔibeR, but not mutant strains ΔibeR or PΔibeR. Lane M, prestained protein marker; Lane 1, ΔibeR (<i>ibeR</i> negative); Lane 2, DE205B (<i>ibeR</i> positive); Lane 3, CΔibeR (<i>ibeR</i> positive); Lane 4, PΔibeR (<i>ibeR</i> negative). <b>(B)</b> Immunoblotting of purified IbeR protein using anti-DE205B. Incubation with anti-DE205B detected protein bands of the expected size for purified IbeR protein. Lane 1, anti-DE205B.</p

Bacterial resistance to environmental stress and SPF chicken serum.

<p><b>(A)</b> Resistance to environmental stress. Each strain was tested for different environmental stress including alkali endurance (pH 10 for 30 min), acid endurance (acetic acid, pH 4.0 and pH 5.0 for 20 min) and high osmolarity challenge (2.4 M NaCl for 1 h). Results were expressed as survival relative to wild-type strain DE205B. Survival of ΔibeR was significantly lower than DE205B (* <i>p <</i> 0.05). The complementation strain CΔibeR recovered the most resistance. <b>(B)</b> Sensitivity to oxidants of DE205B and its ΔibeR and ΔibeA derivatives. Bacterial suspensions were treated with 10 mM H₂O₂ at 37°C for 30 min. After stress exposure, bacteria were diluted in PBS and plated on LB agar. The data were expressed as survival relative to wild-type strain DE205B. Mutant strains ΔibeR, ΔibeA, ΔibeRibeA were more sensitive to H₂O₂ killing than the wild type strain DE205B (** <i>p</i> < 0.01; *** <i>p</i> < 0.001). Moreover, the resistance to H₂O₂ was restored for the complementation strains. <b>(C)</b> Resistance to SPF chicken serum. Bacteria were incubated at 37°C with SPF chicken serum at different dilutions, and counted at 30 min. Mutant strain ΔibeR showed significantly reduced resistance to SPF chicken serum compared to DE205B (* <i>p <</i> 0.05). The error bars indicate standard deviations.</p

Animal infection experiments.

<p><b>(A)</b> Bacterial enumeration during animal systemic infection. Groups of six 8-week-old ICR mice were infected intraperitoneally with a sublethal dose bacterial suspension of 2.0 × 10⁶ CFUs. Bacteria were recovered from blood, brains, lungs, liver and spleen at 24 h post infection. <b>(B)</b> Bacterial enumeration in rat neonatal meningitis model. At 5 days of age, groups of five SPF Sprague-Dawley rat pups were inoculated intraperitoneally with a bacterial suspension containing 10⁷ CFUs. At 18 h after bacterial inoculation, blood and cerebrospinal fluid (CSF) specimens were obtained for quantitative cultures. Nonparametric Mann-Whitney U-Test was carried out for statistical significance analysis. * <i>p</i> < 0.05.</p