Nitric oxide stimulates type IV MSHA pilus retraction in Vibrio cholerae via activation of the phosphodiesterase CdpA

Abstract

Bacteria use surface appendages called type IV pili to perform diverse activities including DNA uptake, twitching motility, and attachment to surfaces. The dynamic extension and retraction of pili are often required for these activities, but the stimuli that regulate these dynamics remain poorly characterized. To address this question, we study the bacterial pathogen Vibrio cholerae, which uses mannose-sensitive hemagglutinin (MSHA) pili to attach to surfaces in aquatic environments as the first step in biofilm formation. Here, we use a combination of genetic and cell biological approaches to describe a regulatory pathway that allows V. cholerae to rapidly abort biofilm formation. Specifically, we show that V. cholerae cells retract MSHA pili and detach from a surface in a diffusion-limited, enclosed environment. This response is dependent on the phosphodiesterase CdpA, which decreases intracellular levels of cyclic-di-GMP to induce MSHA pilus retraction. CdpA contains a putative nitric oxide (NO)–sensing NosP domain, and we demonstrate that NO is necessary and sufficient to stimulate CdpA-dependent detachment. Thus, we hypothesize that the endogenous production of NO (or an NO-like molecule) in V. cholerae stimulates the retraction of MSHA pili. These results extend our understanding of how environmental cues can be integrated into the complex regulatory pathways that control pilus dynamic activity and attachment in bacterial species.

The PilT retraction ATPase promotes both extension and retraction of the MSHA type IVa pilus inVibrio cholerae

“…While these T4aP have distinct extension motor ATPases (MshE for MSHA pili; PilB for competence pili), they both rely on the same ATPase motor, PilT, for efficient retraction ( Fig. 1A-B ), which is consistent with a number of previous studies [4, 22, 23].…”

“…This change in pilus number and length has been previously quantified and shown to differ significantly from the parent strain [23]. These phenotypes have also been qualitatively observed in multiple prior studies [4,15,[22][23][24][25]. While the change in pilus length is visually striking, the major population-level change is the marked reduction in the frequency of piliated cells in the ΔpilT background (Fig.…”

“…1A, C ). This change in pilus number and length has been previously quantified and shown to differ significantly from the parent strain [23]. These phenotypes have also been qualitatively observed in multiple prior studies [4, 15, 22–25].…”

“…1B-C). These results are consistent with multiple prior studies [15,[22][23][24][25]31].One model that has been proposed for the observed reduction in MSHA surface piliation is that PilT is necessary to disrupt pilus extension after it is initiated [25]. Thus, in ΔpilT cells, a single MSHA T4aP machine undergoes uncontrolled "runaway" extension that exhausts the MshA major pilin pool of the cell.…”

“…Conversely, if the retraction motor is disrupted, extension occurs undeterred, resulting in hyperpiliation [5,[16][17][18][19]]. However, these trends are not universally observed, indicating that alternate mechanisms of pilus regulation exist and contribute to surface piliation.Deletion of pilT does not result in hyperpiliation of the T4P in Francisella tularensis [20], Clostridium perfringens [21], or V. cholerae mannose-sensitive hemagglutinin (MSHA) pili [22][23][24][25]. Indeed, for each of these systems, deletion of pilT results in a decrease in piliation.…”