Fig. 5: DspII and DspI synergistically upregulate T3SS gene expression and acute pathogenesis.

A Relative cytotoxicity was assessed by monitoring LDH release from the A549 cells which were infected by the strains of PAO1 WT(EV), ΔdspII(EV), ΔdspI(EV), ΔdspII(dspII), and ΔdspI(dspI). B Acute pathogenesis of PAO1 WT(EV), ΔdspII(EV), ΔdspI(EV), ΔdspII(dspII), and ΔdspI(dspI) strains was measured using the infection model of Galleria mellonella. C Relative expression of exoS, exsA, exsC, and pcrV in the ΔdspII and ΔdspI mutants compared to the PAO1 WT strain was determined by RT-qPCR. D A diagram showing the expression and function of ExsA. ExsA is transcribed from two promoters, PexsCEBA and PexsA, which resulted in polycistronic and monocistronic mRNA, respectively. ExsA positively regulates T3SS genes expression and activates its own transcription through PexsCEBA. E ExsA protein expression in PAO1 WT(EV), ΔdspII(EV), ΔdspI(EV), ΔdspII(dspII), and ΔdspI(dspI) strains was measured using western blot assay. The β-RNA polymerase (β-RNAP) was used as a loading control. Representative blots shown of 2 independent experiments. F, G β-galactosidase activities of the PexsA-lacZ (F) and PexsCEBA-lacZ (G) transcriptional fusion were measured in the PAO1 WT(EV), ΔdspII(EV), ΔdspI(EV), ΔdspII(dspII), and ΔdspI(dspI) strains. (H) β-galactosidase activity of the PexsCEBA-lacZ transcriptional fusion was measured in the PAO1 WT(EV), ΔdspII(EV), ΔdspI(EV), ΔexsA(EV), ΔexsAΔdspII(EV), ΔexsAΔdspII(EV), ΔexsA(exsA), ΔexsAΔdspII(exsA), and ΔexsAΔdspI(exsA) strains. I ExsA protein expression in PAO1 WT(EV), ΔdspII(EV), ΔdspI(EV), ΔexsA(EV), ΔexsAΔdspII(EV), ΔexsAΔdspII(EV), ΔexsA(exsA), ΔexsAΔdspII(exsA), and ΔexsAΔdspI(exsA) strains was measured using western blot assay. Representative blots shown of 2 independent experiments. J β-galactosidase activity of the PexsCEBA-lacZ transcriptional fusion was measured in the PAO1 WT, ΔdspII, ΔdspI, ΔdspIIΔdspI strains and strains with complemented expression of dspII or/and dspI. K β-galactosidase activity of the PexsCEBA-lacZ transcriptional fusion was measured in the PAO1 WT, ΔdspII, and ΔdspII(dspII) strains when they were grown with exogenous addition of CDA at a final concentration of 10 μM, 50 μM, and 100 μM. L β-galactosidase activity of the PexsCEBA-lacZ transcriptional fusion was measured in ΔdspII and ΔdspI mutants with the complementation of their enzymatically dead variants. For (A, C, F–H, J–L) data are presented as mean values  ±  SEM of n = 3 biological replicates. For panel B: data are presented as mean values  ±  SEM of n = 10 biological replicates. Statistical analysis was performed using Student’s t-test (A), one-way ANOVA with Tukey’s multiple comparisons test (F–H, J, L), and two-way ANOVA with Tukey’s multiple comparisons test (C, K). ***, p < 0.0001. Source data are provided as a Source Data file.