c-di-GMP modulates type IV MSHA pilus retraction and surface attachment in Vibrio cholerae

Biofilm formation by Vibrio cholerae facilitates environmental persistence, and hyperinfectivity within the host. Biofilm formation is regulated by 3’,5’-cyclic diguanylate (c-di-GMP) and requires production of the type IV mannose-sensitive hemagglutinin (MSHA) pilus. Here, we show that the MSHA pilus is a dynamic extendable and retractable system, and its activity is directly controlled by c-di-GMP. The interaction between c-di-GMP and the ATPase MshE promotes pilus extension, whereas low levels of c-di-GMP correlate with enhanced retraction. Loss of retraction facilitated by the ATPase PilT increases near-surface roaming motility, and impairs initial surface attachment. However, prolonged retraction upon surface attachment results in reduced MSHA-mediated surface anchoring and increased levels of detachment. Our results indicate that c-di-GMP directly controls MshE activity, thus regulating MSHA pilus extension and retraction dynamics, and modulating V. cholerae surface attachment and colonization.

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Fitnat Yildiz and Gerard Wong 2/20/2020 No custom data collection software not previously described was used for these studies. The source data underlying Figures 2b-d, 2f, 3a-b, 3e, 3g, 4b-e, 7b, Supplementary Figures 1b, 4, 5b-c, 9c, 9e, and 11b are provided as a Source Data file. The source data for all other figures is available upon request from the corresponding authors.
No statistical method was used to determine sample sizes, as sample sizes were chose based upon historical data.
Single-cell experiment sample sizes were determined as previously published in the following references: No data were excluded from these analyses.
All analyses were replicated with at least two biological replicates, or more where indicated, and replication attempts were successful.
There was no randomization for these experiments. All strains were grown under similar conditions, to ensure that bacteria were at equivalent states at the beginning of each experiment. Therefore, all observed differences are attributed to either the genotype differences between the strains being tested, or the variations in the treatment conditions tested on the strains.
Blinding was not used, for as mentioned above, all strains were grown under similar conditions prior to the start of experiments, and the observed differences are attributed to either the genotype or treatment of the strain under the experimental conditions tested.
Rabbit polyclonal anti-MshA was used at a 1:1000 dilution of 1mg/mL stock for immunoblot experiments and 40ug total for Cryo-ET analysis. HRP-conjugated goat anti-rabbit antibody was used for immunoblots at 1:2500 dilution of 2mg/mL stock (Promega Co.)