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.

Thermo-Regulation of the cmaX-cfrX-cmpX Operon IMPORTANCE Because temperature is one of the first physical signal that allows bacteria to distinguish a potential host from its environment, the response of P. aeruginosa has been more investigated in case of elevated rather than low temperature. Here, we show that the ECFσ SigX is an effector in the latter condition, which controls the transcription of the two members of an intriguing operon encoding the mechanosensitive channel CmpX and the hypothetical protein CfrX, while cmaX is under the control of AlgU. In addition, we show that SigX is activated following membrane structure alterations. More generally, we have identified temperature as a key environmental factor regulating antagonistically the expression and the activity of the two major cell wall stress response ECFσ in P. aeruginosa, suggesting that they are involved in specific but interconnected networks, in which the AlgU-dependent global transcriptional regulator AmrZ (alginate and motility regulator Z) could play a role by a mechanism that remains to be explored.

Pseudomonas aeruginosa is an important multidrug-resistant human pathogen by dint of its high intrinsic, acquired, and adaptive resistance mechanisms, causing great concern for immune-compromised individuals and public health. Additionally, P. aeruginosa resilience lies in the production of a myriad of virulence factors, which are known to be tightly regulated by the quorum sensing (QS) system. Anti-virulence therapy has been adopted as an innovative alternative approach to circumvent bacterial antibiotic resistance. Since plants are known repositories of natural phytochemicals, herein, we explored the anti-virulence potential of Azorella atacamensis, a medicinal plant from the Taira Atacama community (Calama, Chile), against P. aeruginosa. Interestingly, A. atacamensis extract (AaE) conferred a significant protection for human lung cells and Caenorhabditis elegans nematodes towards P. aeruginosa pathogenicity. The production of key virulence factors was decreased upon AaE exposure without affecting P. aeruginosa growth. In addition, AaE was able to decrease QS-molecules production. Furthermore, metabolite profiling of AaE and its derived fractions achieved by combination of a molecular network and in silico annotation allowed the putative identification of fourteen diterpenoids bearing a mulinane-like skeleton. Remarkably, this unique interesting group of diterpenoids seems to be responsible for the interference with virulence factors as well as on the perturbation of membrane homeostasis of P. aeruginosa. Hence, there was a significant increase in membrane stiffness, which appears to be modulated by the cell wall stress response ECFσ SigX, an extracytoplasmic function sigma factor involved in membrane homeostasis as well as P. aeruginosa virulence.

30Pseudomonas aeruginosa is capable to deploy a collection of virulence factors that are not 31 only essential for host infection and persistence, but also to escape from the host immune 32 system and to become more resistant to drug therapies. Thus, developing anti-virulence agents 33 that may directly counteract with specific virulence factors or disturb higher regulatory 34 pathways controlling the production of virulence armories are urgently needed. In this regard, 35 this study reports that Pistacia lentiscus L. fruit cyclohexane extract (PLFE1) thwarts P. 36 aeruginosa virulence by targeting mainly the pyocyanin pigment production by interfering with 37 4-hydroxy-2-alkylquinolines molecules production. Importantly, the anti-virulence activity of 38 PLFE1 appears to be associated with membrane homeostasis alteration through the 39 modulation of SigX, an extracytoplasmic function sigma factor involved in cell wall stress 40 response. A thorough chemical analysis of PLFE1 allowed us to identify the ginkgolic acid 41 (C17:1) and hydroginkgolic acid (C15:0) as the main bioactive membrane-interactive 42 compounds responsible for the observed increased membrane stiffness and anti-virulence 43 activity against P. aeruginosa. This study delivers a promising perspective for the potential 44 future use of PLFE1 or ginkgolic acid molecules as an adjuvant therapy to fight against P. 45 aeruginosa infections. 46 47 48 Bacterial infections still constitute a serious public health threat even though their prevention 49 and treatment have been improved over the last decades. The effects of common antibiotics 50 are no longer effective against microbial threats including Enterococcus faecium, 51Staphylococcus aureus, Acinetobacter baumannii, Pseudomonas aeruginosa, and 52Enterobacter species, also known as the "ESKAPE" pathogens group. 1 In a recent report 53 published by the World Health Organization (WHO), P. aeruginosa was categorized as one of 54 the "critical priority pathogens" for which there is an urgent need for the discovery of alternative 55 and innovative new therapies. 2 56 57 P. aeruginosa is predominantly responsible for different life-threatening infections in humans, 58 including the respiratory system, burn and wound, urinary tract as well as medical implant 59 devices. 3 This notorious multidrug resistant opportunistic Gram-negative bacterium deploys a 60 wide variety of virulence factors and host-degrading enzymes as well as multiple secondary 61 metabolites. 4 Pyocyanin is an important virulence factor produced and secreted abundantly by 62 nearly 95% of P. aeruginosa isolates. 5 This phenazine-derived pigment, blue-green in color, 63 confers a greenish hue to the sputum of cystic fibrosis (CF) individuals suffering P. aeruginosa 64 chronic lung infection. 6 Moreover, pyocyanin is a highly diffusible redox-active secondary 65 metabolite which plays an important role in several physiological processes 6,7 making it a good 66 target. Therefore, pyocyanin production hindrance may have consequences regardin...