Fig. 4: Enhanced retraction under low c-di-GMP conditions results from altered MshE activation or functional state.
From: c-di-GMP modulates type IV MSHA pilus retraction and surface attachment in Vibrio cholerae
a Representative overlay images of filtered-fluorescence and phase-contrast channels of mshAT70C strains within surface-associated cells stained with AlexaFluor 488 C5 maleimide dye. Images representative of 3 independent analyses. Scale bars = 2 μm. b Analysis of surface MSHA production via hemagglutination (HA) assay. The reciprocal of the lowest fold dilution at which equivalent cell levels were able to agglutinate sheep erythrocytes (HA Titer) is plotted as the mean with error bars representing the SEM. For each strain n = 5 biological replicates (mshEG11I n = 2), with two technical replicates performed for each biological replicate. WT and Δ4DGC values are the same as shown in Fig. 3e. ND no observable hemagglutination at the highest cell concentration. Statistical analysis: each mutant HA titer was compared to WT via unpaired two-tailed Student’s t-Test, ****p ≤ 0.0001. Source data provided as a Source Data file. c Flagellar motility in soft agar. Data presented as the ratio of mutant motility to WT motility. Graphs depict data as the mean, with error bars representing the standard deviation. n = 6 biological replicates per strain. Statistical analysis: unpaired two-tailed Student’s t-Test, each strain compared to WT ****p ≤ 0.0001, ***p = 0.0001, **p = 0.0012; ###Δ4DGC vs Δ4DGC::mshEL10A/L54A/L58A p = 0.0009, Δ4DGCΔmshE vs Δ4DGC::mshEL10A/L54A/L58A p = 0.0004. d VGJΦ phage transduction levels. All in mshAT70C strain. Individual data points of kanamycin-resistant CFU/mL plotted with line at the mean and error bars representing the standard deviation. Biological replicates: n = 3 per strain. Statistical analysis: unpaired two-tailed Student’s t-Test, ****p ≤ 0.0001. e Planktonic exponential phase c-di-GMP levels determined with the Bc3-5 biosensor. Data presented as mean with standard deviation, n = 3 biological replicates per strain. Statistical analysis, One-way ANOVA compared to WT with Dunnett correction for multiple comparisons, ***p = 0.0002, ****p ≤ 0.0001. Source data provided as a Source Data file.
