The phosphodiesterase NbdA links c-di-GMP signaling to type IV pili function in Pseudomonas aeruginosa PAO1

Scherhag, A.; Aras, K.; Ledermann, M.; Rehner, J.; Karcher, M.; Lang, H.; Stegmuller, S.; Richling, E.; Frankenberg-Dinkel, N.; Zehner, S.

doi:10.64898/2026.03.20.713172

bioRxiv · 10.64898/2026.03.20.713172

The phosphodiesterase NbdA links c-di-GMP signaling to type IV pili function in Pseudomonas aeruginosa PAO1

Scherhag, A.·Aras, K.·Ledermann, M.·Rehner, J.·Karcher, M.·Lang, H.·Stegmuller, S.·Richling, E.·Frankenberg-Dinkel, N.·Zehner, S.

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Abstract

The phosphodiesterase (PDE) NbdA (NO-induced biofilm dispersion locus A) consists of a membrane-integrated MHYT domain, a degenerated diguanylate cyclase (DGC) AGDEF domain and an EAL domain. The integral membrane domain MHYT is proposed to sense a so far unknown extracellular signal and transfers the information to the cytosolic enzyme domains to modulate cellular c-di-GMP level. Here, we show that full length NbdA from Pseudomonas aeruginosa PAO1 is an active PDE in vivo. In line with its PDE activity, overexpression leads to slightly reduced global c-di-GMP levels, and reduced twitching motility. Surprisingly, overexpression of truncated cytosolic NbdA variants exhibited increased c-diGMP levels, suggesting previously uncharacterized DGC activity despite lacking a canonical GGDEF motif. While full-length NbdA overexpression resulted in only slight c-di-GMP reduction, cytosolic variants induced a significant increase, indicating a potential for nonenzymatic effects like protein-protein interactions. Further investigation revealed a connection between NbdA and type IV pilus (T4P) function. Overexpression of NbdA conferred resistance to the T4P-dependent phage DMS3vir, suggesting interference with T4P assembly or function. Microscopic analysis demonstrated dynamic localization of NbdA, partially co-localizing with T4P components, supporting a role in T4P regulation. However, no clear link was re-established with flagellar motor switching or chemotaxis signaling. These findings position NbdA in the complex signaling network of c-diGMP and T4P-mediated surface behavior in P. aeruginosa. Future work will focus on elucidating the precise mechanisms of NbdAs PDE activity and its interplay with other DGC/PDE networks. ImportanceIn this work, we show the in vivo activity of the membrane-bound phosphodiesterase NbdA of Pseudomonas aeruginosa, its role in c-di-GMP homeostasis, cellular localization and implications in surface behavior. Using strains overexpressing NbdA and truncated protein variants, we detected a strong defect in growth on solid surfaces and an altered phage susceptibility. Co-localization experiments supported further the hypothesis of interaction with the type IV pilus apparatus. We propose for NbdA to be part of the protein network responsible for c-di-GMP level modulation at the cell pole and thereby regulating the function of type IV pilus apparatus.

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Scherhag, A., Aras, K., Ledermann, M., Rehner, J., Karcher, M., Lang, H., Stegmuller, S., Richling, E., Frankenberg-Dinkel, N., Zehner, S.. 2026-03-23. The phosphodiesterase NbdA links c-di-GMP signaling to type IV pili function in Pseudomonas aeruginosa PAO1. https://doi.org/10.64898/2026.03.20.713172

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