c-di-GMP-related phenotypes are modulated by the interaction between a diguanylate cyclase and a polar hub protein / Febrero 2020 / Ana Laura Boechat, André A. Pulschen, Caio Gomes Tavares Rosa, Christophe Bordi, Eliezer Stefanello, Gianlucca G. Nicastro, Gilberto H. Kaihami, Jacobo Hernandez-Montelongo, Pio Colepicolo, Regina L. Baldini, Thays De Oliveira Pereira

febrero 20, 2020
Ana Laura Boechat, André A. Pulschen, Caio Gomes Tavares Rosa, Christophe Bordi, Eliezer Stefanello, Gianlucca G. Nicastro, Gilberto H. Kaihami, Jacobo Hernandez-Montelongo, Pio Colepicolo, Regina L. Baldini, Thays de Oliveira Pereira

Abstract:

Over the past decades, (3′-5′)-cyclic diguanylic acid (c-di-GMP) has been characterized as an important second messenger in bacteria. The concentration of c-di-GMP within the cell is associated with cellular behavior: high c-di-GMP levels are linked to biofilm formation and low levels to the motile planktonic lifestyle^1,2. This molecule is synthesized from GTP by a class of enzymes known as diguanylate cyclases (DGC) bearing a conserved GGDEF motif³. The c-di-GMP hydrolysis reaction is performed by phosphodiesterases (PDE) with EAL or HD-GYP domains, which cleave c-di-GMP to pGpG or GMP, respectively^4,5. Multiple genes coding for the c-di-GMP-metabolizing proteins are found in a variety of bacterial genomes. A puzzling question in the study of c-di-GMP signaling is how the bacterial cell integrates the contributions of multiple c-di-GMP-metabolizing enzymes to mediate its cognate functional outcomes. Merritt and collaborators showed that the P. aeruginosa phenotypes controlled by two different DGC have discrete outputs despite the same level of total intracellular c-di-GMP⁶. These data support the model in which localized c-di-GMP signaling contributes to the action of proteins involved in the synthesis, degradation, and/or binding to a downstream target². Studies of c-di-GMP signaling regulation during the swarmer to stalked-cell transition in Caulobacter crescentus also supports this hypothesis. In this dimorphic bacterium, PleD is a DGC that is inactive in swarmer cells and is activated during the swarmer-to-stalked cell differentiation^7,8. Activation of PleD is coupled to its subcellular localization at the stalk pole, suggesting that PleD activates nearby downstream effectors involved in pole remodeling⁹. Opposite to PleD, the EAL domain protein TipF localizes at the swarmer pole, where it contributes to the proper placement of the motor organelle in the polarized predivisional cell¹⁰.

Even though a large body of research on c-di-GMP regulation in P. aeruginosa is available, it is still unclear whether compartmentalization of c-di-GMP signaling components is required to mediate an appropriate c-di-GMP signal transduction. The genome of P. aeruginosa strain PA14 presents forty genes coding for proteins associated with c-di-GMP metabolism^11,12. Some of these proteins were already characterized and a few of them present a specific localization within the cell. For instance, the DGC WspR is associated with contact-dependent response to solid surfaces. Activation of the Wsp system by contact leads to the formation of subcellular clusters of WspR followed by synthesis of c-di-GMP, increasing exopolysaccharide production and biofilm formation¹³. The DGC SadC is a central player in Gac/Rsm-mediated biofilm formation¹⁴ and influences biofilm formation and swarming motility via modulation of exopolysaccharide production and flagellar function¹⁵. It was demonstrated that SadC diguanylate cyclase activity is promoted by membrane association and by direct interaction with flagellar stators disengaged from the motor^16,17. The PDE DipA/Pch is essential for biofilm dispersion¹⁸ and promotes c-di-GMP heterogeneity in P. aeruginosa population¹⁹. This PDE is partitioned after cell division and is localized to the flagellated cell pole by the chemotaxis machinery. This asymmetric distribution during cell division results in a bimodal distribution of c-di-GMP¹⁹.

Previously, we demonstrated that PA14_72420 is an enzymatically active DGC that increased fitness in the presence of sub inhibitory concentrations of imipenem when overproduced in the cells²⁰. Another work referred to this protein as DgcP and indicated its role in virulence²¹, but its molecular function has not yet been addressed. Thus, we decided to pursue its role by seeking for DgcP interaction partners that could participate in the same signaling pathway. DgcP was found to interact with the inner membrane protein FimV, which has a regulatory role in type IV pilus (T4P) function²². Moreover, we determined that DgcP localizes to cell poles in a FimV-dependent manner and is more active when the FimV protein is present. We suggest that the DgcP regulation by FimV may provide a local c-di-GMP pool at the cell pole, making this second messenger available for the c-di-GMP binding proteins that may regulate the machineries associated with the cell motility, such as the flagellum and pili.

https://www.nature.com/articles/s41598-020-59536-9

Ana Laura Boechat, André A. Pulschen, Caio Gomes Tavares Rosa, Christophe Bordi, Eliezer Stefanello, Gianlucca G. Nicastro, Gilberto H. Kaihami, Jacobo Hernandez-Montelongo, Pio Colepicolo, Regina L. Baldini, Thays De Oliveira Pereira

DOI:

c-di-GMP is a major player in the switch between biofilm and motile lifestyles. Several bacteria exhibit a large number of c-di-GMP metabolizing proteins, thus a fine-tuning of this nucleotide levels may occur. It is hypothesized that some c-di-GMP metabolizing proteins would provide the global c-di-GMP levels inside the cell whereas others would maintain a localized pool, with the resulting c-di-GMP acting at the vicinity of its production. Although attractive, this hypothesis has yet to be demonstrated in Pseudomonas aeruginosa. We found that the diguanylate cyclase DgcP interacts with the cytosolic region of FimV, a polar peptidoglycan-binding protein involved in type IV pilus assembly. Moreover, DgcP is located at the cell poles in wild type cells but scattered in the cytoplasm of cells lacking FimV. Overexpression of dgcP leads to the classical phenotypes of high c-di-GMP levels (increased biofilm and impaired motilities) in the wild-type strain, but not in a ΔfimV background. Therefore, our findings suggest that DgcP activity is regulated by FimV. The polar localization of DgcP might contribute to a local c-di-GMP pool that can be sensed by other proteins at the cell pole, bringing to light a specialized function for a specific diguanylate cyclase.

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