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.

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.

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.

MeJA Elicitation of Chicory Hairy Roots Promotes Efficient Increase of 3,5-diCQA Accumulation, a Potent Antioxidant and Antibacterial Molecule.

Cichorium intybus L. (Asteraceae) is an important industrial crop, as well as a medicinal plant which produces some bioactive compounds implicated in various biological effects with potential applications in human health. Particularly, roots produce hydroxycinnamic acids like 5-caffeoyquinic acid and 3,5-dicaffeoylquinic acid (di-CQA). The present investigation relates to the use of methyl jasmonate for enhancing phenolic compounds accumulation and production in hairy root cultures of C. intybus. Elicitated hairy root growth rate increased 13.3 times compared with the initial inoculum in a period of 14 days and di-CQA production represented about 12% of DW. The elicitation has also promoted the production of tricaffeoylquinic acid never described in the chicory roots and identified as 3,4,5-tricaffeoyquinic acid by means of nuclear magnetic resonance. Our study confirmed the strong anti-oxidant effect of di-CQA. Our results also confirmed globally a selectivity of action of di-CQA against Gram-positive bacteria, in particular against some strains of Staphylococcus and Streptococcus. However, a non-negligible antibacterial activity of di-CQA against Pseudomonas aeruginosa was also underlined (MIC = 0.156 mg.mL-1 against some P. aeruginosa strains). The influence of di-CQA has been explored to evaluate its impact on the physiology of P. aeruginosa. Di-CQA showed no effect on the biofilm formation and the production of extracellular pyocyanin. However, it demonstrated an effect on virulence through the production of pyoverdine with a dose-dependent manner by more than 7-fold when treated at a concentration of 128 µg·mL-1, thus suggesting a link between di-CQA and iron sequestration. This study shows that elicitated hairy root cultures of chicory can be developed for the production of di-CQA, a secondary metabolite with high antibacterial potential.

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.