In Pseudomonas aeruginosa, SigX is an extra-cytoplasmic function σ factor that belongs to the cell wall stress response network. In previous studies, we made the puzzling observation that sigX mutant growth was severely affected in rich lysogeny broth (LB) but not in minimal medium. Here, through comparative transcriptomic and proteomic analysis, we show that the absence of SigX results in dysregulation of genes, whose products are mainly involved in transport, carbon and energy metabolisms. Production of most of these genes is controlled by carbon catabolite repression (CCR), a key regulatory system than ensures preferential carbon source uptake and utilization, substrate prioritization and metabolism. The strong CCR response elicited in LB was lowered in a sigX mutant, suggesting altered nutrient uptake. Since the absence of SigX affects membrane composition and fluidity, we suspected membrane changes to cause such phenotype. The detergent polysorbate 80 (PS80) can moderately destabilize the envelope resulting in non-specific increased nutrient intake. Remarkably, growth, membrane fluidity and expression of dysregulated genes in the sigX mutant strain were restored in LB supplemented with PS80. Altogether, these data suggest that SigX is indirectly involved in CCR regulation, possibly via its effects on membrane integrity and fluidity.

Pseudomonas aeruginosa is capable to deploy a collection of virulence factors that are not only essential for host infection and persistence, but also to escape from the host immune system and to become more resistant to drug therapies. Thus, developing anti-virulence agents that may directly counteract with specific virulence factors or disturb higher regulatory pathways controlling the production of virulence armories are urgently needed. In this regard, this study reports that Pistacia lentiscus L. fruit cyclohexane extract (PLFE1) thwarts P. aeruginosa virulence by targeting mainly the pyocyanin pigment production by interfering with 4-hydroxy-2-alkylquinolines molecules production. Importantly, the anti-virulence activity of PLFE1 appears to be associated with membrane homeostasis alteration through the modulation of SigX, an extracytoplasmic function sigma factor involved in cell wall stress response. A thorough chemical analysis of PLFE1 allowed us to identify the ginkgolic acid (C17:1) and hydroginkgolic acid (C15:0) as the main bioactive membrane-interactive compounds responsible for the observed increased membrane stiffness and anti-virulence activity against P. aeruginosa. This study delivers a promising perspective for the potential future use of PLFE1 or ginkgolic acid molecules as an adjuvant therapy to fight against P. aeruginosa infections.

Pseudomonas aeruginosa PAO1 has an integrated Pf4 prophage in its genome, encoding a relatively well-characterized filamentous phage, which contributes to the bacterial biofilm organization and maturation. Pf4 variants are considered as superinfectives when they can re-infect and kill the prophage-carrying host. Herein, the response of P. aeruginosa H103 to Pf4 variant infection was investigated. This phage variant caused partial lysis of the bacterial population and modulated H103 physiology. We show by confocal laser scanning microscopy that a Pf4 variant-infection altered P. aeruginosa H103 biofilm architecture either in static or dynamic conditions. Interestingly, in the latter condition, numerous cells displayed a filamentous morphology, suggesting a link between this phenotype and flow-related forces. In addition, Pf4 variant-infection resulted in cell envelope stress response, mostly mediated by the AlgU and SigX extracytoplasmic function sigma factors (ECFσ). AlgU and SigX involvement may account, at least partly, for the enhanced expression level of genes involved in the biosynthesis pathways of two matrix exopolysaccharides (Pel and alginates) and bis-(3’-5’)-cyclic dimeric guanosine monophosphate (c-di-GMP) metabolism.