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.

Retrospective study on the toxicity induced by stereotactic body radiotherapy: overview of the reunion experience on prostate cancer in elderly patients.

Introduction: Prostate cancer is the fourth most commonly diagnosed cancer among men worldwide. Various tools are used to manage disease such as conventional radiotherapy. However, it has been demonstrated that large prostate volumes were often associated with higher rates of genitourinary and gastrointestinal toxicities. Currently, the improvements in radiotherapy technology have led to the development of stereotactic body radiotherapy, which delivers higher and much more accurate radiation doses. In order to complete literature data about short-term outcome and short-term toxic effects of stereotactic body radiotherapy, we aimed to share our experience about gastrointestinal and genitourinary toxicities associated with stereotactic body radiotherapy in prostate cancer in patients over 70 years old. Methods: We retrospectively reviewed the medical records of elderly patients with prostate cancer treated between 2021 and 2022. The elderly patients were treated with a non-coplanar robotic stereotactic body radiotherapy platform using real-time tracking of implanted fiducials. The prostate, with or without part of the seminal vesicles, was treated with a total dose of 36.25 Gy delivered in five fractions, each fraction being administered every other day. Results: We analyzed a total of 80 elderly patients, comprising 38 low-, 37 intermediate- and 5 high-risk patients. The median follow-up duration was 12 months. We did not observe biochemical/clinical recurrence, distant metastasis, or death. Grade 2 acute genitourinary toxicity was observed in 9 patients (11.25%) and Grade 2 acute gastrointestinal toxicity in 4 patients (5.0%). We did not observe any grade 3 or more acute or late toxicities. Conclusion: Over the follow-up period, we noted a low frequency of gastrointestinal and genitourinary toxicities induced by stereotactic body radiotherapy in the context of prostate cancer in elderly patients. Therefore, stereotactic body radiotherapy seems to represent a promising treatment option for elderly patients, with acceptable acute toxicity.

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.

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.

Deleterious Effects of an Air Pollutant (NO2) on a Selection of Commensal Skin Bacterial Strains, Potential Contributor to Dysbiosis?

The skin constitutes with its microbiota the first line of body defense against exogenous stress including air pollution. Especially in urban or sub-urban areas, it is continuously exposed to many environmental pollutants including gaseous nitrogen dioxide (gNO2). Nowadays, it is well established that air pollution has major effects on the human skin, inducing various diseases often associated with microbial dysbiosis. However, very few is known about the impact of pollutants on skin microbiota. In this study, a new approach was adopted, by considering the alteration of the cutaneous microbiota by air pollutants as an indirect action of the harmful molecules on the skin. The effects of gNO2 on this bacterial skin microbiota was investigated using a device developed to mimic the real-life contact of the gNO2 with bacteria on the surface of the skin. Five strains of human skin commensal bacteria were considered, namely Staphylococcus aureus MFP03, Staphylococcus epidermidis MFP04, Staphylococcus capitis MFP08, Pseudomonas fluorescens MFP05, and Corynebacterium tuberculostearicum CIP102622. Bacteria were exposed to high concentration of gNO2 (10 or 80 ppm) over a short period of 2 h inside the gas exposure device. The physiological, morphological, and molecular responses of the bacteria after the gas exposure were assessed and compared between the different strains and the two gNO2 concentrations. A highly significant deleterious effect of gNO2 was highlighted, particularly for S. capitis MFP08 and C. tuberculostearicum CIP102622, while S. aureus MFP03 seems to be the less sensitive strain. It appeared that the impact of this nitrosative stress differs according to the bacterial species and the gNO2 concentration. Thus the exposition to gNO2 as an air pollutant could contribute to dysbiosis, which would affect skin homeostasis. The response of the microbiota to the nitrosative stress could be involved in some pathologies such as atopic dermatitis.

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.

Activation of the Cell Wall Stress Response in Pseudomonas aeruginosa Infected by a Pf4 Phage Variant.

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.

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.

Membrane-Interactive Compounds From Pistacia lentiscus L. Thwart Pseudomonas aeruginosa Virulence.

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.

Epinephrine affects motility, and increases adhesion, biofilm and virulence of Pseudomonas aeruginosa H103.

Microbial endocrinology has demonstrated for more than two decades, that eukaryotic substances (hormones, neurotransmitters, molecules of the immune system) can modulate the physiological behavior of bacteria. Among them, the hormones/neurotransmitters, epinephrine (Epi) and norepinephrine (NE), released in case of stress, physical effort or used in medical treatment, were shown to be able to modify biofilm formation in various bacterial species. In the present study, we have evaluated the effect of Epi on motility, adhesion, biofilm formation and virulence of Pseudomonas aeruginosa, a bacterium linked to many hospital-acquired infections, and responsible for chronic infection in immunocompromised patients including persons suffering from cystic fibrosis. The results showed that Epi increased adhesion and biofilm formation of P. aeruginosa, as well as its virulence towards the Galleria mellonella larvae in vivo model. Deciphering the sensor of this molecule in P. aeruginosa and the molecular mechanisms involved may help to find new strategies of treatment to fight against this bacterium.

Extracellular DNA release, quorum sensing, and PrrF1/F2 small RNAs are key players in Pseudomonas aeruginosa tobramycin-enhanced biofilm formation.

Biofilms are structured microbial communities that are the leading cause of numerous chronic infections which are difficult to eradicate. Within the lungs of individuals with cystic fibrosis (CF), Pseudomonas aeruginosa causes persistent biofilm infection that is commonly treated with aminoglycoside antibiotics such as tobramycin. However, sublethal concentrations of this aminoglycoside were previously shown to increase biofilm formation by P. aeruginosa, but the underlying adaptive mechanisms still remain elusive. Herein, we combined confocal laser scanning microscope analyses, proteomics profiling, gene expression assays and phenotypic studies to unravel P. aeruginosa potential adaptive mechanisms in response to tobramycin exposure during biofilm growth. Under this condition, we show that the modified biofilm architecture is related at least in part to increased extracellular DNA (eDNA) release, most likely as a result of biofilm cell death. Furthermore, the activity of quorum sensing (QS) systems was increased, leading to higher production of QS signaling molecules. We also demonstrate upon tobramycin exposure an increase in expression of the PrrF small regulatory RNAs, as well as expression of iron uptake systems. Remarkably, biofilm biovolumes and eDNA relative abundances in pqs and prrF mutant strains decrease in the presence of tobramycin. Overall, our findings offer experimental evidences for a potential adaptive mechanism linking PrrF sRNAs, QS signaling, biofilm cell death, eDNA release, and tobramycin-enhanced biofilm formation in P. aeruginosa. These specific adaptive mechanisms should be considered to improve treatment strategies against P. aeruginosa biofilm establishment in CF patients' lungs.

Probiotic Potential and Safety Evaluation of Enterococcus faecalis OB14 and OB15, Isolated From Traditional Tunisian Testouri Cheese and Rigouta, Using Physiological and Genomic Analysis.

Lactic acid bacteria (LAB) strains OB14 and OB15 were isolated from traditional Tunisian fermented dairy products, Testouri cheese and Rigouta, respectively. They were identified as Enterococcus faecalis by the MALDI TOF-MS (matrix assisted laser desorption-ionization time of flight mass spectrometry) biotyper system and molecular assays (species-specific PCR). These new isolates were evaluated for probiotic properties, compared to E. faecalis Symbioflor 1 clone DSM 16431, as reference. The bacteria were found to be tolerant to the harsh conditions of the gastrointestinal tract (acidity and bile salt). They were low to moderate biofilm producers, can adhere to Caco-2/TC7 intestinal cells and strengthen the intestinal barrier through the increase of the transepithelial electrical resistance (TER). Susceptibility to ampicillin, vancomycin, gentamicin and erythromycin has been tested using the broth microdilutions method. The results demonstrated that E. faecalis OB14 and OB15 were sensitive to the clinically important ampicillin (MIC = 1 µg/mL) and vancomycin (MIC = 2 µg/mL) antibiotics. However, Whole Genome Sequencing (WGS) showed the presence of tetracycline resistance and cytolysin genes in E. faecalis OB14, and this led to high mortality of Galleria Mellonella larvae in the virulence test. Hierarchical cluster analysis by MALDI TOF-MS biotyper showed that E. faecalis OB15 was closely related to the E. faecalis Symbioflor 1 probiotic strain than to OB14, and this has been confirmed by WGS using the average nucleotide identity (ANI) and Genome-to-Genome Hybridization similarity methods. According to these results, E. faecalis OB15 seems to be reliable for future development as probiotic, in food or feed industry.

A Flavor Lactone Mimicking AHL Quorum-Sensing Signals Exploits the Broad Affinity of the QsdR Regulator to Stimulate Transcription of the Rhodococcal qsd Operon Involved in Quorum-Quenching and Biocontrol Activities.

In many Gram-negative bacteria, virulence, and social behavior are controlled by quorum-sensing (QS) systems based on the synthesis and perception of N-acyl homoserine lactones (AHLs). Quorum-quenching (QQ) is currently used to disrupt bacterial communication, as a biocontrol strategy for plant crop protection. In this context, the Gram-positive bacterium Rhodococcus erythropolis uses a catabolic pathway to control the virulence of soft-rot pathogens by degrading their AHL signals. This QS signal degradation pathway requires the expression of the qsd operon, encoding the key enzyme QsdA, an intracellular lactonase that can hydrolyze a wide range of substrates. QsdR, a TetR-like family regulator, represses the expression of the qsd operon. During AHL degradation, this repression is released by the binding of the γ-butyrolactone ring of the pathogen signaling molecules to QsdR. We show here that a lactone designed to mimic quorum signals, γ-caprolactone, can act as an effector ligand of QsdR, triggering the synthesis of qsd operon-encoded enzymes. Interaction between γ-caprolactone and QsdR was demonstrated indirectly, by quantitative RT-PCR, molecular docking and transcriptional fusion approaches, and directly, in an electrophoretic mobility shift assay. This broad-affinity regulatory system demonstrates that preventive or curative quenching therapies could be triggered artificially and/or managed in a sustainable way by the addition of γ-caprolactone, a compound better known as cheap food additive. The biostimulation of QQ activity could therefore be used to counteract the lack of consistency observed in some large-scale biocontrol assays.

Mechanism of action of the moonlighting protein EfTu as a Substance P sensor in Bacillus cereus.

The striking feature of the ubiquitous protein EfTu (Thermo unstable ribosomal Elongation factor) is its moonlighting (multifunctional) activity. Beyond its function at the ribosomal level it should be exported to the bacterial surface and act as an environmental sensor. In Bacillus cereus, and other cutaneous bacteria, it serves as a Substance P (SP) receptor and is essential for bacterial adaptation to the host. However, the modus operandi of EfTu as a bacterial sensor remains to be investigated. Studies realized by confocal and transmission electron microscopy revealed that, in the absence of an exogenous signal, EfTu is not exposed on the bacterial surface but is recruited under the effect of SP. In addition, SP acts as a transcriptional regulator of the tuf gene encoding for EfTu. As observed using gadolinium chloride, an inhibitor of membrane mechanosensitive channels (Msc), Msc control EfTu export and subsequently the bacterial response to SP both in terms of cytotoxicity and biofilm formation activity. Microscale thermophoresis revealed that in response to SP, EfTu can form homopolymers. This event should occur after EfTu export and, as shown by proteo-liposome reconstruction studies, SP appears to promote EfTu polymers association to the membrane, leading subsequently to the bacterial response. Molecular modeling suggests that this mechanism should involve EfTu unfolding and insertion into the bacterial cytoplasmic membrane, presumably through formation of homopolymers. This study is unraveling the original mechanism action of EfTu as a bacterial sensor but also reveals that this protein should have a broader role, including in eukaryotes.

Extracytoplasmic function sigma factors in Pseudomonas aeruginosa.

The opportunistic pathogen Pseudomonas aeruginosa, like all members of the genus Pseudomonas, has the capacity to thrive in very different environments, ranging from water, plant roots, to animals, including humans to whom it can cause severe infections. This remarkable adaptability is reflected in the number of transcriptional regulators, including sigma factors in this bacterium. Among those, the 19 to 21 extracytoplasmic sigma factors (ECFσ) are endowed with different regulons and functions, including the iron starvation σ (PvdS, FpvI, HasI, FecI, FecI2 and others), the cell wall stress ECFσ AlgU, SigX and SbrI, and the unorthodox σVreI involved in the expression of virulence. Recently published data show that these ECFσ have separate regulons although presenting some cross-talk. We will present evidence that these different ECFσ are involved in the expression of different phenotypes, ranging from cell-wall stress response, production of extracellular polysaccharides, formation of biofilms, to iron acquisition.

A Rhodococcal Transcriptional Regulatory Mechanism Detects the Common Lactone Ring of AHL Quorum-Sensing Signals and Triggers the Quorum-Quenching Response.

The biocontrol agent Rhodococcus erythropolis disrupts virulence of plant and human Gram-negative pathogens by catabolizing their N-acyl-homoserine lactones. This quorum-quenching activity requires the expression of the qsd (quorum-sensing signal degradation) operon, which encodes the lactonase QsdA and the fatty acyl-CoA ligase QsdC, involved in the catabolism of lactone ring and acyl chain moieties of signaling molecules, respectively. Here, we demonstrate the regulation of qsd operon expression by a TetR-like family repressor, QsdR. This repression was lifted by adding the pathogen quorum signal or by deleting the qsdR gene, resulting in enhanced lactone degrading activity. Using interactomic approaches and transcriptional fusion strategy, the qsd operon derepression was elucidated: it is operated by the binding of the common part of signaling molecules, the homoserine lactone ring, to the effector-receiving domain of QsdR, preventing a physical binding of QsdR to the qsd promoter region. To our knowledge, this is the first evidence revealing quorum signals as inducers of the suitable quorum-quenching pathway, confirming this TetR-like protein as a lactone sensor. This regulatory mechanism designates the qsd operon as encoding a global disrupting pathway for degrading a wide range of signal substrates, allowing a broad spectrum anti-virulence activity mediated by the rhodococcal biocontrol agent. Understanding the regulation mechanisms of qsd operon expression led also to the development of biosensors useful to monitor in situ the presence of exogenous signals and quorum-quenching activity.

The absence of SigX results in impaired carbon metabolism and membrane fluidity in Pseudomonas aeruginosa.

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.

In vitro Assessment of the Probiotic Properties and Bacteriocinogenic Potential of Pediococcus pentosaceus MZF16 Isolated From Artisanal Tunisian Meat "Dried Ossban".

Pediococcus pentosaceus MZF16 has been isolated from artisanal Tunisian meat so called "Dried Ossban," an original ecological niche, and identified by MALDI-TOF mass spectrometry and 16S rDNA sequencing. This bacterium showed a high tolerance to gastric stress conditions, and toward bile salts. P. pentosaceus MZF16 also demonstrated a hydrophobic surface profile (high adhesion to xylene), autoaggregation, and adhesive abilities to the human intestinal Caco-2/TC7 cell line. These properties may help the bacterium colonizing the gut. Furthermore, MZF16 was found to be resistant to gentamycin and chloramphenicol but did not harbor any transferable resistance determinants and/or virulence genes. The data also demonstrated absence of cytotoxicity of this strain. Conversely, P. pentosaceus MZF16 can slightly stimulate the immune system and enhance the intestinal epithelial barrier function. Moreover, this bacterium has been shown to be highly active against Listeria spp. due to bacteriocin production. Characterization of the bacteriocin by PCR amplification, sequencing and bioinformatic analyses revealed that MZF16 produces a bacteriocin 100% identical to coagulin, a pediocin-like inhibitory substance produced by Bacillus coagulans. To our knowledge, this is the first report that highlights the production of a pediocin 100% identical to coagulin in a Pediococcus strain. As coagulin, pediocin MZF16 has the consensus sequence YYGNGVXCXXXXCXVXXXXA (X denotes any amino acid), which confirms its belonging to class IIa bacteriocins, and its suitability to preserve foods from Listeria monocytogenes development. According to these results, P. pentosaceus MZF16 can be proposed as a probiotic and bioprotective agent for fermented foods, including Tunisian dry meat and sausages. Further investigations will aim to study the behavior of this strain in meat products as a component of functional food.