Phosphatidylserine externalization and procoagulant activation of erythrocytes induced by Pseudomonas aeruginosa virulence factor pyocyanin

The opportunistic pathogen Pseudomonas aeruginosa causes a wide range of infections in multiple hosts by releasing an arsenal of virulence factors such as pyocyanin. Despite numerous reports on the pleiotropic cellular targets of pyocyanin toxicity in vivo, its impact on erythrocytes remains elusive. Erythrocytes undergo an apoptosis‐like cell death called eryptosis which is characterized by cell shrinkage and phosphatidylserine (PS) externalization; this process confers a procoagulant phenotype on erythrocytes as well as fosters their phagocytosis and subsequent clearance from the circulation. Herein, we demonstrate that P. aeruginosa pyocyanin‐elicited PS exposure and cell shrinkage in erythrocyte while preserving the membrane integrity. Mechanistically, exposure of erythrocytes to pyocyanin showed increased cytosolic Ca²⁺ activity as well as Ca²⁺‐dependent proteolytic processing of μ‐calpain. Pyocyanin further up‐regulated erythrocyte ceramide abundance and triggered the production of reactive oxygen species. Pyocyanin‐induced increased PS externalization in erythrocytes translated into enhanced prothrombin activation and fibrin generation in plasma. As judged by carboxyfluorescein succinimidyl‐ester labelling, pyocyanin‐treated erythrocytes were cleared faster from the murine circulation as compared to untreated erythrocytes. Furthermore, erythrocytes incubated in plasma from patients with P. aeruginosa sepsis showed increased PS exposure as compared to erythrocytes incubated in plasma from healthy donors. In conclusion, the present study discloses the eryptosis‐inducing effect of the virulence factor pyocyanin, thereby shedding light on a potentially important mechanism in the systemic complications of P. aeruginosa infection.     

Fitness trade‐offs explain low levels of persister cells in the opportunistic pathogen Pseudomonas aeruginosa

Microbial populations often contain a fraction of slow‐growing persister cells that withstand antibiotics and other stress factors. Current theoretical models predict that persistence levels should reflect a stable state in which the survival advantage of persisters under adverse conditions is balanced with the direct growth cost impaired under favourable growth conditions, caused by the nonreplication of persister cells. Based on this direct growth cost alone, however, it remains challenging to explain the observed low levels of persistence (<<1%) seen in the populations of many species. Here, we present data from the opportunistic human pathogen Pseudomonas aeruginosa that can explain this discrepancy by revealing various previously unknown costs of persistence. In particular, we show that in the absence of antibiotic stress, increased persistence is traded off against a lengthened lag phase as well as a reduced survival ability during stationary phase. We argue that these pleiotropic costs contribute to the very low proportions of persister cells observed among natural P. aeruginosa isolates (3 × 10^?8–3 × 10^?4) and that they can explain why strains with higher proportions of persister cells lose out very quickly in competition assays under favourable growth conditions, despite a negligible difference in maximal growth rate. We discuss how incorporating these trade‐offs could lead to models that can better explain the evolution of persistence in nature and facilitate the rational design of alternative therapeutic strategies for treating infectious diseases.     

Co‐evolutionary dynamics between public good producers and cheats in the bacterium Pseudomonas aeruginosa

The production of beneficial public goods is common in the microbial world, and so is cheating – the exploitation of public goods by nonproducing mutants. Here, we examine co‐evolutionary dynamics between cooperators and cheats and ask whether cooperators can evolve strategies to reduce the burden of exploitation, and whether cheats in turn can improve their exploitation abilities. We evolved cooperators of the bacterium Pseudomonas aeruginosa, producing the shareable iron‐scavenging siderophore pyoverdine, together with cheats, defective in pyoverdine production but proficient in uptake. We found that cooperators managed to co‐exist with cheats in 56% of all replicates over approximately 150 generations of experimental evolution. Growth and competition assays revealed that co‐existence was fostered by a combination of general adaptions to the media and specific adaptions to the co‐evolving opponent. Phenotypic screening and whole‐genome resequencing of evolved clones confirmed this pattern, and suggest that cooperators became less exploitable by cheats because they significantly reduced their pyoverdine investment. Cheats, meanwhile, improved exploitation efficiency through mutations blocking the costly pyoverdine‐signalling pathway. Moreover, cooperators and cheats evolved reduced motility, a pattern that likely represents adaptation to laboratory conditions, but at the same time also affects social interactions by reducing strain mixing and pyoverdine sharing. Overall, we observed parallel evolution, where co‐existence of cooperators and cheats was enabled by a combination of adaptations to the abiotic and social environment and their interactions.     

Cross‐Species Alleviation of Biotic and Abiotic Stresses by the Endophyte Pseudomonas aeruginosa PW09

A wheat endophytic bacterium (Pseudomonas aeruginosa PW09) was evaluated for its ability to trigger an induced systemic resistance response in cucumber against biotic and abiotic stresses. PW09 was applied to cucumber seeds, and the seedlings were subjected to Sclerotium rolfsii infection and NaCl (150 mm ). The role of PW09 was evaluated in alleviating the stresses by assessing plant mortality due to S. rolfsii infection and biomass accumulation under NaCl stress as well as at the physiological level through phenylpropanoid metabolism, antioxidant activities and proline accumulation. The endophyte reduced seedling mortality by 60% and increased biomass accumulation significantly under S. rolfsii (7%) and NaCl (18%) stresses, respectively, compared with endophyte‐untreated seedlings. Application of PW09 also induced higher accumulation of proline (1.3‐ and 1.4‐fold) and total phenolics (1.2‐ and 1.1‐fold) and activities of polyphenol oxidase (4.3‐ and 1.5‐fold), phenylalanine ammonia lyase (1.29‐ and 1.27‐fold) and superoxide dismutase (2.5‐ and 1.39‐fold) under S. rolfsii and NaCl stresses, indicating the ability of the wheat endophyte PW09 in alleviating both biotic and abiotic stresses in cucumber.     

Sequential interactions of silver–silica nanocomposite (Ag–SiO2NC) with cell wall,metabolism and genetic stability of Pseudomonas aeruginosa,a multiple antibiotic‐resistant bacterium

The study was carried out to understand the effect of silver–silica nanocomposite (Ag–SiO₂NC) on the cell wall integrity, metabolism and genetic stability of Pseudomonas aeruginosa, a multiple drug‐resistant bacterium. Bacterial sensitivity towards antibiotics and Ag–SiO₂NC was studied using standard disc diffusion and death rate assay, respectively. The effect of Ag–SiO₂NC on cell wall integrity was monitored using SDS assay and fatty acid profile analysis, while the effect on metabolism and genetic stability was assayed microscopically, using CTC viability staining and comet assay, respectively. Pseudomonas aeruginosa was found to be resistant to β‐lactamase, glycopeptidase, sulfonamide, quinolones, nitrofurantoin and macrolides classes of antibiotics. Complete mortality of the bacterium was achieved with 80 μg ml^?1 concentration of Ag–SiO₂NC. The cell wall integrity reduced with increasing time and reached a plateau of 70% in 110 min. Changes were also noticed in the proportion of fatty acids after the treatment. Inside the cytoplasm, a complete inhibition of electron transport system was achieved with 100 μg ml^?1 Ag–SiO₂NC, followed by DNA breakage. The study thus demonstrates that Ag–SiO₂NC invades the cytoplasm of the multiple drug‐resistant P. aeruginosa by impinging upon the cell wall integrity and kills the cells by interfering with electron transport chain and the genetic stability.

Significance and Impact of Study

Although the synthesis, structural characteristics and biofunction of silver nanoparticles are well understood, their application in antimicrobial therapy is still at its infancy as only a small number of microorganisms are tested to be sensitive to nanoparticles. A thorough knowledge of the mode of interaction of nanoparticles with bacteria at subcellular level is mandatory for any clinical application. The present study deals with the interactions of Ag–SiO2NC with the cell wall integrity, metabolism and genetic stability of Pseudomonas aeruginosa, which would contribute substantially in strengthening the therapeutic applications of silver nanoparticles.