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

Recherche de nouveaux actifs d'origine végétale contre le pathogène opportuniste de l'homme Pseudomonas aeruginosa

Antimicrobial resistance has become a great challenge in therapeutic medicine so much so that the World health organization forecasts the possibility of a post-antibiotic era where minor injuries may lead to mortality. Pseudomonas aeruginosa is among the list of organisms that are highly resistant to conventional antibiotics, partly due to its broad genome, which facilitates the elaboration of virulence determinants and rapid adaptation to various environments, in addition to its inherent resistance mechanisms. In view of this, alternative measures of controlling microbial virulence activities using novel approaches that do not disturb its growth and viability, also known as anti-virulence strategy, are gaining wider attention. Since plants are repositories of several metabolites with chemical defense system against environmental pathogens, through ethnobotanical led studies, the effect of Pistacia lentiscus fruit extracts originating from Algeria and forty plant extracts originating from North-Chile were biologically and chemically evaluated with the aim of deciphering their anti-virulence effects against P. aeruginosa. Furthermore, this study tried to gain more insight into the bioactive compounds and possible mechanism of action. From the results obtained, selected plant extracts attenuated P. aeruginosa mainly pyocyanin activity and /or elastase and rhamnolipids virulence production which appears to be associated with the inhibition of quorum sensing activities and the alteration in membrane activities. The anti-virulence effect of the selected extracts (P. lentiscus, Azorella atacamensis, Baccharis grisebachii, Haplopappus rigidus and Parastrephia terestiucula) were also validated in biological models of infections where they mediated the toxicity of P. aeruginosa towards A549 human monolayer cells and/or Caenorhabditis elegans nematode. Interestingly, growth of the pathogen was not affected. Further chemical profiling of P. Lentiscus, and A atacamensis extracts revealed the presence of gingkolic acid and azorellane/mulinane diterpenoids as the putative bioactive compounds. Future studies intend to explore these extracts and their derived compounds on the potentiation of antibiotic activity in a panel of clinical strains. In general, this study sets the pace for the possible use of these plant extracts as adjuvants in treatment of P. aeruginosa infections.La résistance aux antimicrobiens est l’un des défis majeurs du XX1eme siècle. Pseudomonas aeruginosa est inscrit sur la liste des organismes pathogènes qui deviennent résistants aux antibiotiques conventionnels. De nouvelles stratégies visant à atténuer la virulence sans perturber la croissance et la viabilité bactériennes, également connues sous le nom de stratégie anti-virulence, sont développées. Les plantes sont connues pour produire de nombreux métabolites secondaires. Des extraits de fruits de Pistachia Lentiscus originaires d'Algérie et de 40 extraits de plantes originaires du Nord-Chili ont été criblés pour leur capacité à atténuer la production de la pyocyanine, un facteur de virulence majeur de P. aeruginosa, dans le but d’évaluer leur potentiel effet antivirulence. Les extraits sélectionnés (Pistacia lentiscus, Azorella atacamensis, Baccharis grisebachii, Haplopappus rigidus et Parastrephia terestiucula), ont été fractionnés et l’ensemble de ces extraits et fractions a montré une atténuation de la production d’autres facteurs de virulence (élastase, rhamnolipides), qui a pu être attribuée, au moins partiellement à une diminution de la communication bactérienne via le mécanisme du quorum sensing. Ces extraits et fractions altèrent également la fluidité membranaire de P. aeruginosa. Cet effet anti-virulence a été validé dans un modèle d'infection cellulaire, et sur le nématode Caenorhabditis elegans. Dans toutes ces conditions, la croissance de P. aeruginosa n'a pas été affectée. Un profilage chimique des extraits et fractions de P. lentiscus et d'A atacamensis a révélé la présence d'acide gingkolique et de diterpenoides de type azorellane/mulinane comme potentiels composés bioactifs. De futures études visent à identifier les composés bioactifs sur P. aeruginosa H103, ainsi que sur un panel de souches cliniques, et à évaluer un potentiel effet potentialisateur de l'activité des antibiotiques. Ces travaux visent in fine à proposer ces composés d’origine végétale comme adjuvants dans le traitement des infections à P. aeruginosa

Searching for new bioactive compounds to tackle the opportunistic human pathogen Pseudomonas aeruginosa

La résistance aux antimicrobiens est l’un des défis majeurs du XX1eme siècle. Pseudomonas aeruginosa est inscrit sur la liste des organismes pathogènes qui deviennent résistants aux antibiotiques conventionnels. De nouvelles stratégies visant à atténuer la virulence sans perturber la croissance et la viabilité bactériennes, également connues sous le nom de stratégie anti-virulence, sont développées. Les plantes sont connues pour produire de nombreux métabolites secondaires. Des extraits de fruits de Pistachia Lentiscus originaires d'Algérie et de 40 extraits de plantes originaires du Nord-Chili ont été criblés pour leur capacité à atténuer la production de la pyocyanine, un facteur de virulence majeur de P. aeruginosa, dans le but d’évaluer leur potentiel effet antivirulence. Les extraits sélectionnés (Pistacia lentiscus, Azorella atacamensis, Baccharis grisebachii, Haplopappus rigidus et Parastrephia terestiucula), ont été fractionnés et l’ensemble de ces extraits et fractions a montré une atténuation de la production d’autres facteurs de virulence (élastase, rhamnolipides), qui a pu être attribuée, au moins partiellement à une diminution de la communication bactérienne via le mécanisme du quorum sensing. Ces extraits et fractions altèrent également la fluidité membranaire de P. aeruginosa. Cet effet anti-virulence a été validé dans un modèle d'infection cellulaire, et sur le nématode Caenorhabditis elegans. Dans toutes ces conditions, la croissance de P. aeruginosa n'a pas été affectée. Un profilage chimique des extraits et fractions de P. lentiscus et d'A atacamensis a révélé la présence d'acide gingkolique et de diterpenoides de type azorellane/mulinane comme potentiels composés bioactifs. De futures études visent à identifier les composés bioactifs sur P. aeruginosa H103, ainsi que sur un panel de souches cliniques, et à évaluer un potentiel effet potentialisateur de l'activité des antibiotiques. Ces travaux visent in fine à proposer ces composés d’origine végétale comme adjuvants dans le traitement des infections à P. aeruginosa.Antimicrobial resistance has become a great challenge in therapeutic medicine so much so that the World health organization forecasts the possibility of a post-antibiotic era where minor injuries may lead to mortality. Pseudomonas aeruginosa is among the list of organisms that are highly resistant to conventional antibiotics, partly due to its broad genome, which facilitates the elaboration of virulence determinants and rapid adaptation to various environments, in addition to its inherent resistance mechanisms. In view of this, alternative measures of controlling microbial virulence activities using novel approaches that do not disturb its growth and viability, also known as anti-virulence strategy, are gaining wider attention. Since plants are repositories of several metabolites with chemical defense system against environmental pathogens, through ethnobotanical led studies, the effect of Pistacia lentiscus fruit extracts originating from Algeria and forty plant extracts originating from North-Chile were biologically and chemically evaluated with the aim of deciphering their anti-virulence effects against P. aeruginosa. Furthermore, this study tried to gain more insight into the bioactive compounds and possible mechanism of action. From the results obtained, selected plant extracts attenuated P. aeruginosa mainly pyocyanin activity and /or elastase and rhamnolipids virulence production which appears to be associated with the inhibition of quorum sensing activities and the alteration in membrane activities. The anti-virulence effect of the selected extracts (P. lentiscus, Azorella atacamensis, Baccharis grisebachii, Haplopappus rigidus and Parastrephia terestiucula) were also validated in biological models of infections where they mediated the toxicity of P. aeruginosa towards A549 human monolayer cells and/or Caenorhabditis elegans nematode. Interestingly, growth of the pathogen was not affected. Further chemical profiling of P. Lentiscus, and A atacamensis extracts revealed the presence of gingkolic acid and azorellane/mulinane diterpenoids as the putative bioactive compounds. Future studies intend to explore these extracts and their derived compounds on the potentiation of antibiotic activity in a panel of clinical strains. In general, this study sets the pace for the possible use of these plant extracts as adjuvants in treatment of P. aeruginosa infections

Role Of CmpX in membrane fluidity, pyocyanin production and biofilm formation in Pseudomonas aeruginosa H103

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

Abstract 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