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Nitric oxide stimulates type IV MSHA pilus retraction in Vibrio cholerae via activation of the phosphodiesterase CdpA
Saturday, 2022/02/19 | 06:14:04

Hannah Q. Hughes, Kyle A. Floyd, Sajjad Hossain, Sweta Anantharaman, David T. Kysela, Miklόs Zöldi, Lászlό Barna, Yuanchen Yu, Michael P. Kappler, Triana N. Dalia, Ram C. Podicheti, Douglas B. Rusch, Meng Zhuang, Cassandra L. Fraser, Yves V. Brun, Stephen C. Jacobson, James B. McKinlay, Fitnat H. Yildiz, Elizabeth M. Boon, and Ankur B. Dalia

 

PNAS February 15, 2022 119 (7) e2108349119

Significance

All organisms sense and respond to their environments. One way bacteria interact with their surroundings is by dynamically extending and retracting filamentous appendages from their surface called pili. While pili are critical for many functions, such as attachment, motility, and DNA uptake, the factors that regulate their dynamic activity are poorly understood. Here, we describe how an environmental signal induces a signaling pathway to promote the retraction of mannose-sensitive hemagglutinin pili in Vibrio cholerae. The retraction of these pili promotes the detachment of V. cholerae from a surface and may provide a means by which V. cholerae can respond to changes in its environment.

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.

 

See https://www.pnas.org/content/119/7/e2108349119

 

Fig. 1. Cells rapidly detach from the surface of a glass well slide in a manner that is dependent on MSHA pilus retraction. (A) Representative montage illustrating that MSHA pilus retraction precedes cell detachment (Movie S1). Phase images (Top) show cell boundaries, and FITC channel (Bottom) images show AF488-mal labeled MSHA pili. White arrows indicate examples where pili retract prior to cell detachment. There are 10-s intervals between frames. Scale bar, 1 μm. (B) Diagram of experimental setup to observe cell detachment in a glass well slide (Top). Representative phase contrast images (Bottom) of cell detachment over time. There is a 1-min interval between frames. Scale bar, 10 μm. (C and D) MixD assays of the indicated strains. Cells expressed GFP (green) or mCherry (fuchsia) as indicated by the color-coded genotypes. Representative montages show time-lapse imaging (Left) with 1-min intervals between frames (Movies S2 and S3). Scale bar, 10 μm. The quantification of three biological replicates is shown in the line graph (Right) and is displayed as the mean ± SD. Statistical comparisons were made by one-way ANOVA and post hoc Holm-Šídák test. *P < 0.05, ***P < 0.001, ****P < 0.0001. (E) Representative montage shows the labeled fluorescent MSHA pili in a mixture of a pilT-mCherry strain and a CyPet-expressing parent strain (Movie S4). Phase contrast images with overlaid CyPet fluorescence in cyan (Top) distinguish the two strains, and FITC fluorescence images in green (Bottom) show AF488-mal labeled pili. Parent cells are outlined in cyan and pilT-mCherry cells are outlined in white. There are 20-s intervals between frames. Scale bar, 1μm. The PilT-mCherry construct does not exhibit detectable mCherry fluorescence in D and E.

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