cmpX overexpression in Pseudomonas aeruginosa affects biofilm formation and cell morphology in response to shear stress.

Pseudomonas aeruginosa is an opportunistic pathogen causing chronic infections that are related to its ability to form biofilms. Mechanosensitive ion channels (Mcs) are cytoplasmic membrane proteins whose opening depends on a mechanical stress impacting the lipid bilayer. CmpX is a homologue of the small conductance MscS of Escherichia coli. The cmpX gene is part of a transcriptional cfrX-cmpX unit that is under the control of the cell envelope stress response ECF sigma factor SigX. CmpX was shown to regulate the activity of the hybrid sensor kinase PA1611 involved in the regulation of transition from a planktonic to a biofilm lifestyle. The deletion of cmpX leads to increased biofilm formation under static conditions. Herein, the effect of cmpX overexpression was investigated by confocal laser scanning microscopy in terms of biofilm formation and architecture, and matrix components production, in dynamic conditions. We show that overexpression of cmpX in P. aeruginosa leads to enhanced and altered biofilm architecture that seems to be associated to increased matrix components and the emergence of filamentous cells. These phenotypic alterations might occur potentially through a shear stress induced by the medium flow rate. Importance: CmpX is involved in biofilm formation and cell filamentation with regards to the medium flow.

Membrane fluidity homeostasis is required for tobramycin-enhanced biofilm in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic pathogen, which causes chronic infections, especially in cystic fibrosis (CF) patients where it colonizes the lungs via the build-up of biofilms. Tobramycin, an aminoglycoside, is often used to treat P. aeruginosa infections in CF patients. Tobramycin at sub-minimal inhibitory concentrations enhances both biofilm biomass and thickness in vitro; however, the mechanism(s) involved are still unknown. Herein, we show that tobramycin increases the expression and activity of SigX, an extracytoplasmic sigma factor known to be involved in the biosynthesis of membrane lipids and membrane fluidity homeostasis. The biofilm enhancement by tobramycin is not observed in a sigX mutant, and the sigX mutant displays increased membrane stiffness. Remarkably, the addition of polysorbate 80 increases membrane fluidity of sigX-mutant cells in biofilm, restoring the tobramycin-enhanced biofilm formation. Our results suggest the involvement of membrane fluidity homeostasis in biofilm development upon tobramycin exposure.IMPORTANCEPrevious studies have shown that sub-lethal concentrations of tobramycin led to an increase biofilm formation in the case of infections with the opportunistic pathogen Pseudomonas aeruginosa. We show that the mechanism involved in this phenotype relies on the cell envelope stress response, triggered by the extracytoplasmic sigma factor SigX. This phenotype was abolished in a sigX-mutant strain. Remarkably, we show that increasing the membrane fluidity of the mutant strain is sufficient to restore the effect of tobramycin. Altogether, our data suggest the involvement of membrane fluidity homeostasis in biofilm development upon tobramycin exposure.

Sensitivity of Legionella pneumophila to phthalates and their substitutes.

Phthalates constitute a family of anthropogenic chemicals developed to be used in the manufacture of plastics, solvents, and personal care products. Their dispersion and accumulation in many environments can occur at all stages of their use (from synthesis to recycling). However, many phthalates together with other accumulated engineered chemicals have been shown to interfere with hormone activities. These compounds are also in close contact with microorganisms that are free-living, in biofilms or in microbiota, within multicellular organisms. Herein, the activity of several phthalates and their substitutes were investigated on the opportunistic pathogen Legionella pneumophila, an aquatic microbe that can infect humans. Beside showing the toxicity of some phthalates, data suggested that Acetyl tributyl citrate (ATBC) and DBP (Di-n-butyl phthalate) at environmental doses (i.e. 10-6 M and 10-8 M) can modulate Legionella behavior in terms of motility, biofilm formation and response to antibiotics. A dose of 10-6 M mostly induced adverse effects for the bacteria, in contrast to a dose of 10-8 M. No perturbation of virulence towards Acanthamoeba castellanii was recorded. These behavioral alterations suggest that L. pneumophila is able to sense ATBC and DBP, in a cross-talk that either mimics the response to a native ligand, or dysregulates its physiology.

N-Acylsulfonamide: a valuable moiety to design new sulfa drug analogues.

Sulfonamides are the oldest class of antibiotics, discovered more than 80 years ago. They are still used today despite the appearance of drug resistance phenomena that limit their prescription. Since the discovery and use of the first sulfa drugs, many analogues have been synthesized in order to obtain new active molecules able to circumvent bacterial resistance. Structurally similar to sulfonamide, the N-acylsulfonamide group arouses interest in the field of medicinal chemistry due to specific physico-chemical properties. We report here the synthesis and antibacterial/antibiofilm activities of 18 sulfa drug analogues with an N-acylsulfonamide moiety. These derivatives were obtained efficiently by sulfo-click reactions between readily available thioacid and sulfonyl azide synthons.

The natriuretic peptide receptor agonist osteocrin disperses Pseudomonas aeruginosa biofilm.

Biofilms are highly tolerant to antimicrobials and host immune defense, enabling pathogens to thrive in hostile environments. The diversity of microbial biofilm infections requires alternative and complex treatment strategies. In a previous work we demonstrated that the human Atrial Natriuretic Peptide (hANP) displays a strong anti-biofilm activity toward Pseudomonas aeruginosa and that the binding of hANP by the AmiC protein supports this effect. This AmiC sensor has been identified as an analog of the human natriuretic peptide receptor subtype C (h-NPRC). In the present study, we evaluated the anti-biofilm activity of the h-NPRC agonist, osteocrin (OSTN), a hormone that displays a strong affinity for the AmiC sensor at least in vitro. Using molecular docking, we identified a pocket in the AmiC sensor that OSTN reproducibly docks into, suggesting that OSTN might possess an anti-biofilm activity as well as hANP. This hypothesis was validated since we observed that OSTN dispersed established biofilm of P. aeruginosa PA14 strain at the same concentrations as hANP. However, the OSTN dispersal effect is less marked than that observed for the hANP (-61% versus -73%). We demonstrated that the co-exposure of P. aeruginosa preformed biofilm to hANP and OSTN induced a biofilm dispersion with a similar effect to that observed with hANP alone suggesting a similar mechanism of action of these two peptides. This was confirmed by the observation that OSTN anti-biofilm activity requires the activation of the complex composed by the sensor AmiC and the regulator AmiR of the ami pathway. Using a panel of both P. aeruginosa laboratory reference strains and clinical isolates, we observed that the OSTN capacity to disperse established biofilms is highly variable from one strain to another. Taken together, these results show that similarly to the hANP hormone, OSTN has a strong potential to be used as a tool to disperse P. aeruginosa biofilms.

High affinity iron uptake by pyoverdine in Pseudomonas aeruginosa involves multiple regulators besides Fur, PvdS, and FpvI.

Pseudomonas aeruginosa is a Gram-negative bacterium which can cause serious infections among immune-depressed people including cystic fibrosis patients where it can colonize the lungs causing chronic infections. Iron is essential for P. aeruginosa and can be provided via three sources under aerobic conditions: its own siderophores pyochelin (PCH) and pyoverdine (PVD), xenosiderophores, or heme, respectively. Pyoverdine is the high affinity siderophore and its synthesis and uptake involve more than 30 genes organized in different operons. Its synthesis and uptake are triggered by iron scarcity via the Fur regulator and involves two extra cytoplasmic sigma factors (ECF), PvdS for the biosynthesis of PVD and FpvI for the uptake via the TonB-dependent FpvA outer membrane transporter and other periplasmic and inner membrane proteins. It appeared recently that the regulation of PVD biosynthesis and uptake involves other regulators, including other ECF factors, and LysR regulators. This is the case especially for the genes coding for periplasmic and inner membrane proteins involved in the reduction of Fe3+ to Fe2+ and the transport of ferrous iron to the cytoplasm that appears to represent a crucial step in the uptake process.

Antibiofilm and Antivirulence Properties of 6-Polyaminosteroid Derivatives against Antibiotic-Resistant Bacteria.

The emergence of multi-drug resistant pathogens is a major public health problem, leading us to rethink and innovate our bacterial control strategies. Here, we explore the antibiofilm and antivirulence activities of nineteen 6-polyaminosterol derivatives (squalamine-based), presenting a modulation of their polyamine side chain on four major pathogens, i.e., carbapenem-resistant A. baumannii (CRAB) and P. aeruginosa (CRPA), methicillin-resistant S. aureus (MRSA), and vancomycin-resistant E. faecium (VRE) strains. We screened the effect of these derivatives on biofilm formation and eradication. Derivatives 4e (for CRAB, VRE, and MRSA) and 4f (for all the strains) were the most potent ones and displayed activities as good as those of conventional antibiotics. We also identified 11 compounds able to decrease by more than 40% the production of pyocyanin, a major virulence factor of P. aeruginosa. We demonstrated that 4f treatment acts against bacterial infections in Galleria mellonella and significantly prolonged larvae survival (from 50% to 80%) after 24 h of CRAB, VRE, and MRSA infections. As shown by proteomic studies, 4f triggered distinct cellular responses depending on the bacterial species but essentially linked to cell envelope. Its interesting antibiofilm and antivirulence properties make it a promising a candidate for use in therapeutics.

Organs-on-Chips Platforms Are Everywhere: A Zoom on Biomedical Investigation.

Over the decades, conventional in vitro culture systems and animal models have been used to study physiology, nutrient or drug metabolisms including mechanical and physiopathological aspects. However, there is an urgent need for Integrated Testing Strategies (ITS) and more sophisticated platforms and devices to approach the real complexity of human physiology and provide reliable extrapolations for clinical investigations and personalized medicine. Organ-on-a-chip (OOC), also known as a microphysiological system, is a state-of-the-art microfluidic cell culture technology that sums up cells or tissue-to-tissue interfaces, fluid flows, mechanical cues, and organ-level physiology, and it has been developed to fill the gap between in vitro experimental models and human pathophysiology. The wide range of OOC platforms involves the miniaturization of cell culture systems and enables a variety of novel experimental techniques. These range from modeling the independent effects of biophysical forces on cells to screening novel drugs in multi-organ microphysiological systems, all within microscale devices. As in living biosystems, the development of vascular structure is the salient feature common to almost all organ-on-a-chip platforms. Herein, we provide a snapshot of this fast-evolving sophisticated technology. We will review cutting-edge developments and advances in the OOC realm, discussing current applications in the biomedical field with a detailed description of how this technology has enabled the reconstruction of complex multi-scale and multifunctional matrices and platforms (at the cellular and tissular levels) leading to an acute understanding of the physiopathological features of human ailments and infections in vitro.

Cell Envelope Stress Response in Pseudomonas aeruginosa.

Bacteria sense their environment via the cell envelope, which in Gram-negative bacteria comprises the outer membrane, the periplasmic space, and the inner membrane. Pseudomonas aeruginosa is an opportunistic pathogen which is exposed to different cell wall stresses imposed by exposure to antibiotics, osmotic pressure, and long-time colonization of host tissues such as the lung in cystic fibrosis patients. In response to these stresses, P. aeruginosa is able to respond by establishing a cell envelope stress response involving different regulatory pathways including the extra-cytoplasmic sigma factors AlgU, SigX, and SbrI and other two-component sensor/response regulators and effectors. This chapter aims to review the different factors leading to the activation of the cell envelope stress response in P. aeruginosa and the genetic determinants involved in this response, which is crucial for the survival of the bacterium upon exposure to different stressful conditions.

Effect of Phthalates and Their Substitutes on the Physiology of Pseudomonas aeruginosa.

Phthalates are used in a variety of applications-for example, as plasticizers in polyvinylchloride products to improve their flexibility-and can be easily released into the environment. In addition to being major persistent organic environmental pollutants, some phthalates are responsible for the carcinogenicity, teratogenicity, and endocrine disruption that are notably affecting steroidogenesis in mammals. Numerous studies have thus focused on deciphering their effects on mammals and eukaryotic cells. While multicellular organisms such as humans are known to display various microbiota, including all of the microorganisms that may be commensal, symbiotic, or pathogenic, few studies have aimed at investigating the relationships between phthalates and bacteria, notably regarding their effects on opportunistic pathogens and the severity of the associated pathologies. Herein, the effects of phthalates and their substitutes were investigated on the human pathogen, Pseudomonas aeruginosa, in terms of physiology, virulence, susceptibility to antibiotics, and ability to form biofilms. We show in particular that most of these compounds increased biofilm formation, while some of them enhanced the bacterial membrane fluidity and altered the bacterial morphology.

Pf4 Phage Variant Infection Reduces Virulence-Associated Traits in Pseudomonas aeruginosa.

Pf4 is a filamentous bacteriophage integrated as a prophage into the genome of Pseudomonas aeruginosa PAO1. Pf4 virions can be produced without killing P. aeruginosa. However, cell lysis can occur during superinfection when Pf virions successfully infect a host lysogenized by a Pf superinfective variant. We have previously shown that infection of P. aeruginosa PAO1 with a superinfective Pf4 variant abolished twitching motility and altered biofilm architecture. More precisely, most of the cells embedded into the biofilm were showing a filamentous morphology, suggesting the activation of the cell envelope stress response involving both AlgU and SigX extracytoplasmic function sigma factors. Here, we show that Pf4 variant infection results in a drastic dysregulation of 3,360 genes representing about 58% of P. aeruginosa genome; of these, 70% of the virulence factors encoding genes show a dysregulation. Accordingly, Pf4 variant infection (termed Pf4*) causes in vivo reduction of P. aeruginosa virulence and decreased production of N-acyl-homoserine lactones and 2-alkyl-4-quinolones quorum-sensing molecules and related virulence factors, such as pyocyanin, elastase, and pyoverdine. In addition, the expression of genes involved in metabolism, including energy generation and iron homeostasis, was affected, suggesting further relationships between virulence and central metabolism. Altogether, these data show that Pf4 phage variant infection results in complex network dysregulation, leading to reducing acute virulence in P. aeruginosa. This study contributes to the comprehension of the bacterial response to filamentous phage infection. IMPORTANCE Filamentous bacteriophages can become superinfective and infect P. aeruginosa, even though they are inserted in the genome as lysogens. Despite this productive infection, growth of the host is only mildly affected, allowing the study of the interaction between the phage and the host, which is not possible in the case of lytic phages killing rapidly their host. Here, we demonstrate by transcriptome and phenotypic analysis that the infection by a superinfective filamentous phage variant causes a massive disruption in gene expression, including those coding for virulence factors and metabolic pathways.

Gaseous NO2 induces various envelope alterations in Pseudomonas fluorescens MFAF76a.

Anthropogenic atmospheric pollution and immune response regularly expose bacteria to toxic nitrogen oxides such as NO• and NO2. These reactive molecules can damage a wide variety of biomolecules such as DNA, proteins and lipids. Several components of the bacterial envelope are susceptible to be damaged by reactive nitrogen species. Furthermore, the hydrophobic core of the membranes favors the reactivity of nitrogen oxides with other molecules, making membranes an important factor in the chemistry of nitrosative stress. Since bacteria are often exposed to endogenous or exogenous nitrogen oxides, they have acquired protection mechanisms against the deleterious effects of these molecules. By exposing bacteria to gaseous NO2, this work aims to analyze the physiological effects of NO2 on the cell envelope of the airborne bacterium Pseudomonas fluorescens MFAF76a and its potential adaptive responses. Electron microscopy showed that exposure to NO2 leads to morphological alterations of the cell envelope. Furthermore, the proteomic profiling data revealed that these cell envelope alterations might be partly explained by modifications of the synthesis pathways of multiple cell envelope components, such as peptidoglycan, lipid A, and phospholipids. Together these results provide important insights into the potential adaptive responses to NO2 exposure in P. fluorescens MFAF76a needing further investigations.

Impact of Carbon Source Supplementations on Pseudomonas aeruginosa Physiology.

Pseudomonas aeruginosa is an opportunistic pathogen highly resistant to a wide range of antimicrobial agents, making its infections very difficult to treat. Since microorganisms need to perpetually adapt to their surrounding environment, understanding the effect of carbon sources on P. aeruginosa physiology is therefore essential to avoid increasing drug-resistance and better fight this pathogen. By a global proteomic approach and phenotypic assays, we investigated the impact of various carbon source supplementations (glucose, glutamate, succinate, and citrate) on the physiology of the P. aeruginosa PA14 strain. A total of 581 proteins were identified as differentially expressed in the 4 conditions. Most of them were more abundant in citrate supplementation and were involved in virulence, motility, biofilm development, and antibiotic resistance. Phenotypic assays were performed to check these hypotheses. By coupling all this data, we highlight the importance of the environment in which the bacterium evolves on its metabolism, and thus the necessity to better understand the metabolic pathways implied in its adaptative response according to the nutrient availability.

Pseudomonas aeruginosa Biofilm Dispersion by the Human Atrial Natriuretic Peptide.

Pseudomonas aeruginosa biofilms cause chronic, antibiotic tolerant infections in wounds and lungs. Numerous recent studies demonstrate that bacteria can detect human communication compounds through specific sensor/receptor tools that modulate bacterial physiology. Consequently, interfering with these mechanisms offers an exciting opportunity to directly affect the infection process. It is shown that the human hormone Atrial Natriuretic Peptide (hANP) both prevents the formation of P. aeruginosa biofilms and strongly disperses established P. aeruginosa biofilms. This hANP action is dose-dependent with a strong effect at low nanomolar concentrations and takes effect in 30-120 min. Furthermore, although hANP has no antimicrobial effect, it acts as an antibiotic adjuvant. hANP enhances the antibiofilm action of antibiotics with diverse modes of action, allowing almost full biofilm eradication. The hANP effect requires the presence of the P. aeruginosa sensor AmiC and the AmiR antiterminator regulator, indicating a specific mode of action. These data establish the activation of the ami pathway as a potential mechanism for P. aeruginosa biofilm dispersion. hANP appears to be devoid of toxicity, does not enhance bacterial pathogenicity, and acts synergistically with antibiotics. These data show that hANP is a promising powerful antibiofilm weapon against established P. aeruginosa biofilms in chronic infections.

Pharmacological validation of Solanum mammosum L. as an anti-infective agent: Role of solamargine.

ETHNOPHARMACOLOGICAL RELEVANCE: Fungal and bacterial infections remain a major problem worldwide, requiring the development of effective therapeutic strategies. Solanum mammosum L. (Solanaceae) ("teta de vaca") is used in traditional medicine in Peru to treat fungal infections and respiratory disorders via topical application. However, the mechanism of action remains unknown, particularly in light of its chemical composition. MATERIALS AND METHODS: The antifungal activity of TDV was determined against Trichophyton mentagrophytes and Candida albicans using bioautography-TLC-HRMS to rapidly identify the active compounds. Then, the minimum inhibitory concentration (MIC) of the fruit crude extract and the active compound was determined to precisely evaluate the antifungal activity. Additionally, the effects of the most active compound on the formation of Pseudomonas aeruginosa biofilms and pyocyanin production were evaluated. Finally, a LC-HRMS profile and a molecular network of TDV extract were created to characterize the metabolites in the fruits' ethanolic extract. RESULTS: Bioautography-TLC-HRMS followed by isolation and confirmation of the structure of the active compound by 1D and 2D NMR allowed the identification solamargine as the main compound responsible for the anti-Trichophyton mentagrophytes (MIC = 64 µg mL-1) and anti-Candida albicans (MIC = 64 µg mL-1) activities. In addition, solamargine led to a significant reduction of about 20% of the Pseudomonas aeruginosa biofilm formation. This effect was observed at a very low concentration (1.6 µg mL-1) and remained fairly consistent regardless of the concentration. In addition, solamargine reduced pyocyanin production by about 20% at concentrations of 12.5 and 50 µg mL-1. Furthermore, the LC-HRMS profiling of TDV allowed us to annotate seven known compounds that were analyzed through a molecular network. CONCLUSIONS: Solamargine has been shown to be the most active compound against T. mentoagrophytes and C. albicans in vitro. In addition, our data show that this compound affects significantly P. aeruginosa pyocyanin production and biofilm formation in our conditions. Altogether, these results might explain the traditional use of S. mammosum fruits to treat a variety of fungal infections and respiratory disorders.

Variability of the response of human vaginal Lactobacillus crispatus to 17ß-estradiol.

We previously showed that the physiological concentration of 17ß-estradiol in the vaginal environment is sufficient to affect the membrane dynamics and adhesion phenotype of the Lactobacillus crispatus strain CIP104459. However, L. crispatus is a heterogeneous species. Here, we investigated the effect of 17ß-estradiol on the recently isolated L. crispatus vaginal strain V4, related to a cluster distant from CIP104459 and at the limit of being a different subspecies. Grown in the same medium, the two strains expressed a highly similar pool of proteins. However, in contrast to CIP104459, L. crispatus V4 showed high aggregation potential and 17ß-estradiol promoted this phenotype. This effect was associated with large changes in cell-surface polarity and Lewis acid/base properties. In addition, we observed no effect on the membrane dynamics, contrary to CIP104459. These results can be explained by differences in the properties and organization of the S layer between the two strains. However, as for CIP104459, 17ß-estradiol increased biosurfactant production of L. crispatus V4 and their adhesion to vaginal cells. This suggests that 17ß-estradiol agonists would be valuable tools to favor a stable re-implantation of L. crispatus in the vaginal mucosa.

Effect of 17ß-estradiol on a human vaginal Lactobacillus crispatus strain.

Lactobacilli and estrogens play essential roles in vaginal homeostasis. We investigated the potential direct effect of 17ß-estradiol on a vaginal strain of Lactobacillus crispatus, the major bacterial species of the vaginal microbiota. 17ß-estradiol (10-6 to 10-10 M) had no effect on L. crispatus growth, but markedly affected the membrane dynamics of this bacterium. This effect appeared consistent with a signal transduction process. The surface polarity and aggregation potential of the bacterium were unaffected by exposure to 17ß-estradiol, but its mean size was significantly reduced. 17ß-estradiol also promoted biosurfactant production by L. crispatus and adhesion to vaginal VK2/E6E7 cells, but had little effect on bacterial biofilm formation activity. Bioinformatic analysis of L. crispatus identified a membrane lipid raft-associated stomatin/prohibitin/flotillin/HflK domain containing protein as a potential 17ß-estradiol binding site. Overall, our results reveal direct effects of 17ß-estradiol on L. crispatus. These effects are of potential importance in the physiology of the vaginal environment, through the promotion of lactobacillus adhesion to the mucosa and protection against pathogens.

MacAB-TolC Contributes to the Development of Acinetobacter baumannii Biofilm at the Solid-Liquid Interface.

Acinetobacter baumannii has emerged as one of the most problematic bacterial pathogens responsible for hospital-acquired and community infections worldwide. Besides its high capacity to acquire antibiotic resistance mechanisms, it also presents high adhesion abilities on inert and living surfaces leading to biofilm development. This lifestyle confers additional protection against various treatments and allows it to persist for long periods in various hospital niches. Due to their remarkable antimicrobial tolerance, A. baumannii biofilms are difficult to control and ultimately eradicate. Further insights into the mechanism of biofilm development will help to overcome this challenge and to develop novel antibiofilm strategies. To unravel critical determinants of this sessile lifestyle, the proteomic profiles of two A. baumannii strains (ATTC17978 and SDF) grown in planktonic stationary phase or in mature solid-liquid (S-L) biofilm were compared using a semiquantitative proteomic study. Of interest, among the 69 common proteins determinants accumulated in the two strains at the S-L interface, we sorted out the MacAB-TolC system. This tripartite efflux pump played a role in A. baumannii biofilm formation as demonstrated by using ΔmacAB-tolC deletion mutant. Complementary approaches allowed us to get an overview of the impact of macAB-tolC deletion in A. baumannii physiology. Indeed, this efflux pump appeared to be involved in the envelope stress response occurring in mature biofilm. It contributes to maintain wild type (WT) membrane rigidity and provides tolerance to high osmolarity conditions. In addition, this system is probably involved in the maintenance of iron and sulfur homeostasis. MacAB-TolC might help this pathogen face and adapt to deleterious conditions occurring in mature biofilms. Increasing our knowledge of A. baumannii biofilm formation will undoubtedly help us develop new therapeutic strategies to tackle this emerging threat to human health.

Tackling Pseudomonas aeruginosa Virulence by Mulinane-Like Diterpenoids from Azorella atacamensis.

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.

The Temperature-Regulation of Pseudomonas aeruginosa cmaX-cfrX-cmpX Operon Reveals an Intriguing Molecular Network Involving the Sigma Factors AlgU and SigX.

Pseudomonas aeruginosa is a highly adaptable Gram-negative opportunistic pathogen, notably due to its large number of transcription regulators. The extracytoplasmic sigma factor (ECFσ) AlgU, responsible for alginate biosynthesis, is also involved in responses to cell wall stress and heat shock via the RpoH alternative σ factor. The SigX ECFσ emerged as a major regulator involved in the envelope stress response via membrane remodeling, virulence and biofilm formation. However, their functional interactions to coordinate the envelope homeostasis in response to environmental variations remain to be determined. The regulation of the putative cmaX-cfrX-cmpX operon located directly upstream sigX was investigated by applying sudden temperature shifts from 37°C. We identified a SigX- and an AlgU- dependent promoter region upstream of cfrX and cmaX, respectively. We show that cmaX expression is increased upon heat shock through an AlgU-dependent but RpoH independent mechanism. In addition, the ECFσ SigX is activated in response to valinomycin, an agent altering the membrane structure, and up-regulates cfrX-cmpX transcription in response to cold shock. Altogether, these data provide new insights into the regulation exerted by SigX and networks that are involved in maintaining envelope homeostasis.