Fig. 2: MSHA are dynamic retractile pili, and retraction is requisite upon the PilT ATPase.
From: c-di-GMP modulates type IV MSHA pilus retraction and surface attachment in Vibrio cholerae
a Time-lapse visualization of MSHA retraction in surface-associated mshAT70C cells labeled with AlexaFluor 594 C5 maleimide. Images were collected every 6 seconds over 10 min. White arrow in all images marks the tip of the retracting pilus at t = 0. Numerous retraction events were observed over multiple biological replicates (see Supplementary Movies). Scale bar = 2 μm. b Transduction of VGJΦ is dependent upon production of MSHA pili. Individual data points plotted with line at the mean and error bars representing the standard deviation. Biological replicates: WT n = 8, ΔmshA n = 6, ΔmshE n = 6, ΔpilT n = 6, ΔpilU n = 6, ΔpilTΔpilU n = 6. Statistical analysis: unpaired two-tailed Student’s t-Test, **p = 0.0024. ND no detected kanamycin-resistant colonies. Data to the right of Dashed-line: WT and mshAT70C cells treated with 4.2 mM MeOH-PEG-mal in DMSO prior to introduction of VGJΦ. Individual data points plotted with line at the mean and error bars representing the standard deviation. Biological replicates: WT n = 4, mshAT70C n = 6. Statistical analysis, unpaired two-tailed Students t-Test, ****p ≤ 0.0001. Source data provided as a Source Data file. c Average force of MSHA retraction determined by micropillars assay. Individual data points plotted with line at mean and error bars representing the standard deviation. ND no retraction events detected. Biological replicates: WT n = 17 and ΔpilU n = 16. Statistical analysis of WT vs. ΔpilU, unpaired two-tailed Students t-Test, not significant p = 0.5291. Source data provided as a Source Data file. d Average speed of MSHA retraction determined by micropillar assay. Individual data points plotted with line at mean and error bars representing the standard deviation. ND no retraction events detected. Biological replicates: WT n = 17ΔpilU n = 16. Statistical analysis of WT vs. ΔpilU, unpaired two-tailed Students t-Test, not significant p = 0.1428. Source data provided as a Source Data file. e Representative overlay images of filtered-fluorescence and phase-contrast channels from ATPase mutants in the mshAT70C strain within surface-associated cells stained with AlexaFluor 488 C5 maleimide dye. Images representative of three independent analyses. Scale bars = 2 μm. f 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 were analyzed, with two technical replicates performed for each biological replicate. 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.
