Giovanni Bertoni

Biofilm formation in Pseudomonas aeruginosa is regulated by intersecting signals from the envelope stress response and the cyclic di-GMP (c-di-GMP) second messenger network. We previously described that the envelope stress response sigma factor σ22 can simultaneously drive transcriptional activation and, via the small RNA ErsA, post-transcriptional repression of algC, which encodes a central enzyme in the production of sugar precursors for biofilm matrix exopolysaccharides. The inhibitory effect of ErsA on AlgC appeared incongruent with the well-established role of high c-di-GMP levels in promoting exopolysaccharide synthesis. In this study, we address this apparent contradiction by demonstrating that elevated c-di-GMP levels relieve ErsA-mediated repression of AlgC. This regulatory mechanism also applies to other ErsA targets, including the envelope homeostasis lipoprotein MlaA and the flagellar regulator FleN, which cooperates with FleQ to promote the biosynthesis of Pel/Psl exopolysaccharides and the CdrAB adhesins under high c-di-GMP conditions. Collectively, our results support a model in which ErsA serves as a central, c-di-GMP-responsive regulatory hub, linking envelope stress responses to c-di-GMP cascades to fine-tune key extracytoplasmic functions and coordinate biofilm formation.

Background: The carbapenem antibiotic meropenem is often used to treat life-threatening infections caused by Pseudomonas aeruginosa . Previous studies have shown that the susceptibility of P. aeruginosa to carbapenems is differentially regulated by the RNA chaperone Hfq, depending on the availability of preferred or less preferred carbon sources, a mechanism known as carbon catabolite repression (CCR). In this regulation, Hfq plays a CCR-conditioned repressive role on outer membrane porins that act as entry ports for carbapenems. In this study, we investigated whether meropenem response is modulated by the second messenger c-di-GMP, which is known to regulate several bacterial functions. Methods: We used P. aeruginosa strains with high or low c-di-GMP levels and their Hfq-deficient derivatives to assess the role of c-di-GMP in modulating meropenem susceptibility and tolerance. Results: We show that low intracellular c−di−GMP levels increase meropenem resistance and tolerance at sub−inhibitory concentrations, whereas high c−di−GMP diminishes both traits. Importantly, c−di−GMP status shapes the entire response trajectory, from exponential growth to the stationary phase. Furthermore, we show that c-di-GMP modulates meropenem response through mechanism(s) independent of Hfq-mediated porin repression and exerts a dominant effect over CCR-driven regulation. Conclusion: This study supports the notion that P. aeruginosa meropenem susceptibility and tolerance are modulated by intracellular c-di-GMP concentrations, with low c-di-GMP levels promoting higher fitness. Our findings indicate that c-di-GMP exerts its regulatory effect through mechanisms distinct from Hfq-mediated porin control, underscoring the existence of parallel regulatory pathways that shape antibiotic response.

Protein–protein interactions (PPIs) have been recognized as a promising target for the development of new drugs, as proved by the growing number of PPI modulators reaching clinical trials. In this context, peptides represent a valid alternative to small molecules, owing to their unique ability to mimic the target protein structure and interact with wider surface areas. Among the possible fields of interest, bacterial PPIs represent an attractive target to face the urgent necessity to fight antibiotic resistance. Growing attention has been paid to the YgjD/YeaZ/YjeE complex responsible for the essential t⁶A₃₇ tRNA modification in bacteria. We previously identified an α‐helix on the surface of Pseudomonas aeruginosa YeaZ, crucial for the YeaZ‐YeaZ homodimer formation and the conserved YeaZ‐YgjD interactions. Herein, we present our studies for impairing the PPIs involved in the formation of the YeaZ dimers through synthetic peptide derivatives of this helical moiety, both in vitro with purified components and on P. aeruginosa cells. Our results proved the possibility of targeting those PPIs which are usually essential for protein functioning and thus are refractory to mutational changes and antibiotic resistance development.

In recent years, several genome-wide approaches based on RNA sequencing (RNA-seq) have been developed. These methods allow a comprehensive and dynamic view of the structure and function of the multi-layered RNA pathways and networks. Many of these approaches, including the promising one of single-cell transcriptome analysis, have been successfully applied to Pseudomonas aeruginosa. However, we are only at the beginning because only a few surrounding conditions have been considered. Here, we aim to illustrate the different types of approaches based on RNA-seq that will lead us in the future to a better understanding of the dynamics of RNA biology in P. aeruginosa. Key words Pseudomonas aeruginosa RNA-seq Small RNAs RNA binding protein Transcriptional control Post-transcriptional control RNA-RNA interaction RNA-protein interaction

Several animal models have been developed to study infection strategies and pathogenesis of Pseudomonas aeruginosa. Some of these models are also used in preclinical and clinical research. However, these models are increasingly showing their limitations, including in recapitulating human diseases such as cystic fibrosis, which is strongly linked to P. aeruginosa infection. The emerging field of human organoids and organs-on-a-chip is expected to provide answers to the need for in vitro modeling of human diseases. Here, we describe the first recent efforts that will hopefully provide the basis for the development of advanced in vitro models for the study of P. aeruginosa infection and pathogenesis and the testing of antibacterial agents. Key words Pseudomonas aeruginosa Cystic fibrosis Organoids Organs-on-chips Animal models Infection model

Hundreds of different species of small RNAs can populate a bacterial cell. This small transcriptome contains important information for the adaptation of cellular physiology to environmental changes. Underlying cellular networks involving small RNAs are RNA–RNA and RNA-protein interactions, which are often intertwined. In addition, small RNAs can function as mRNAs. In general, small RNAs are referred to as noncoding because very few are known to contain translated open reading frames. In this article, we intend to highlight that the number of small RNAs that fall within the set of translated RNAs is bound to increase. In addition, we aim to emphasize that the dynamics of the small transcriptome involve different functional codes, not just the genetic code. Therefore, since the role of small RNAs is always code-driven, we believe that there is little reason to continue calling them small noncoding RNAs.

The pathogenic lifestyle of the bacterium Pseudomonas aeruginosa , a leading cause of life-threatening infections in the airways of cystic fibrosis patients, is based on the fine regulation of virulence-associated factors. Regulatory small RNAs (sRNAs) and the RNA-binding protein Hfq are recognized key components within the P. aeruginosa regulatory networks involved in host-pathogen interaction.

CFTR modulators have been developed to correct and/or enhance CFTR activity in patients with specific cystic fibrosis (CF) genotypes. However, it is of great importance to identify potential off-targets of these novel therapies to understand how they affect lung physiology in CF.

Cystic fibrosis transmembrane conductance regulator (CFTR) modulators improve clinical outcomes with variable efficacy in patients with cystic fibrosis (CF). However, changes produced by bacterial persistence and adaptation in addition to antibiotic regimens could influence CFTR modulator efficacy and vice versa and hence clinical outcomes. We first evaluated the effects of ivacaftor (IVA), lumacaftor (LUM), tezacaftor, elexacaftor and elexacaftor/tezacaftor/ivacaftor (ETI), alone or combined with antibiotics, on sequential Staphylococcus aureus and Pseudomonas aeruginosa CF isolates. IVA and ETI showed the most potent direct antimicrobial activity against S. aureus , while P. aeruginosa was not affected. Additive effects or synergies were observed between the CFTR modulators and antibiotics against both S. aureus and P. aeruginosa , independently of the stage of colonization. IVA and LUM were the most effective in potentiating antibiotic activity against S. aureus , while IVA and ETI enhanced mainly polymyxins activity against P. aeruginosa . Next, we evaluated the effect of P. aeruginosa pneumonia on the pharmacokinetics of IVA in mice. The time-concentration curves of IVA and its metabolites in plasma, lung and epithelial lining fluid were influenced by P. aeruginosa infection. The area under the concentration-time curve showed that airway exposure to IVA was greater in infected than non-infected mice. These results suggest that CFTR modulators can have direct antimicrobial properties and/or enhance antibiotic activity against S. aureus and P. aeruginosa . Furthermore, bacterial infection impacts the IVA concentration and airway exposure, potentially affecting its efficacy. Our findings suggest optimizing host- and pathogen-directed drug regimens to improve efficacy for personalized treatment.