Figure 4.

Differential expression profile of Bpm in the absence of a T6SS, reveals a role for BicA as a potential virulence factor associated with IEC infection. A. Volcano plot of the 200 most differentially expressed genes associated with Bpm IEC infection generated with genes with a p-values less than 0.05. The plot shows dysregulation of gene expression associated with two important mechanisms of virulence, namely, T3SS and T6SS. B. Using a transposon library, several mutants dysregulated during IEC infection were tested for their ability to form plaques. C. A ΔbicA mutant and a complemented ΔbicA::bicA were generated and tested for plaque assays (MOI 0.1 or 1) at 24 h. D. IECs were infected with Bpm WT, ΔbicA or ΔbicA::bicA for 1 h using an MOI of 10. After infection, extracellular bacteria were killed using 1 mg/mL of kanamycin for 1 h. Intracellular bacterial replication was quantified by CFU enumeration at 3, 6, 12 and 24 h. Error bars of the mean represent the average ± standard error of two experiments with three biological replicates. Statistical analysis was done using a one-way ANOVA followed by a Sidak multiple comparison test. *p < .05, **p < .01, ****p < .0001, ns, not significant. E. Primary IECs were infected with Bpm WT, ΔbicA or ΔbicA::bicA for 6 or 12 h at 37°C and 5% CO₂, using an MOI of 10. After infection, cells were washed with PBS, fixed, and stained, prior to microscopic examination (20×). Bacterial cells were stained with sera collected from mice that immunized with Bpm PBK001 live attenuated vaccine, followed by an Alexa fluorophore 488 goat anti-mouse IgG, IgM, IgA (H + L) secondary antibody. Actin was stained using rhodamine-phalloidin and cell nuclei with DAPI. Magnified views (×5) are shown on the bottom panels. Images were processed and analyzed using ImageJ analysis software.