Dataset Information

Pseudomonas aeruginosa N-3-oxo-dodecanoyl-homoserine Lactone Elicits Changes in Cell Volume, Morphology, and AQP9 Characteristics in Macrophages.

ABSTRACT: Quorum sensing (QS) communication allows Pseudomonas aeruginosa to collectively control its population density and the production of biofilms and virulence factors. QS signal molecules, like N-3-oxo-dodecanoyl-L-homoserine lactone (3O-C12-HSL), can also affect the behavior of host cells, e.g., by modulating the chemotaxis, migration, and phagocytosis of human leukocytes. Moreover, host water homeostasis and water channels aquaporins (AQP) are critical for cell morphology and functions as AQP interact indirectly with the cell cytoskeleton and signaling cascades. Here, we investigated how P. aeruginosa 3O-C12-HSL affects cell morphology, area, volume and AQP9 expression and distribution in human primary macrophages, using quantitative PCR, immunoblotting, two- and three-dimensional live imaging, confocal and nanoscale imaging. Thus, 3O-C12-HSL enhanced cell volume and area and induced cell shape and protrusion fluctuations in macrophages, processes tentatively driven by fluxes of water across cell membrane through AQP9, the predominant AQP in macrophages. Moreover, 3O-C12-HSL upregulated the expression of AQP9 at both the protein and mRNA levels. This was accompanied with enhanced whole cell AQP9 fluorescent intensity and redistribution of AQP9 to the leading and trailing regions, in parallel with increased cell area in the macrophages. Finally, nanoscopy imaging provided details on AQP9 dynamics and architecture within the lamellipodial area of 3O-C12-HSL-stimulated cells. We suggest that these novel events in the interaction between P. aeruginosa and macrophage may have an impact on the effectiveness of innate immune cells to fight bacteria, and thereby resolve the early stages of infections and inflammations.

SUBMITTER: Holm A

PROVIDER: S-EPMC4805602 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Pseudomonas aeruginosa N-3-oxo-dodecanoyl-homoserine Lactone Elicits Changes in Cell Volume, Morphology, and AQP9 Characteristics in Macrophages.

Holm Angelika A Magnusson Karl-Eric KE Vikström Elena E

Frontiers in cellular and infection microbiology 20160324

Quorum sensing (QS) communication allows Pseudomonas aeruginosa to collectively control its population density and the production of biofilms and virulence factors. QS signal molecules, like N-3-oxo-dodecanoyl-L-homoserine lactone (3O-C12-HSL), can also affect the behavior of host cells, e.g., by modulating the chemotaxis, migration, and phagocytosis of human leukocytes. Moreover, host water homeostasis and water channels aquaporins (AQP) are critical for cell morphology and functions as AQP int ...[more]

PMID: 27047801

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Project description:Bacterial quorum sensing (QS) is a cell-to-cell communication system that governs the expression of a large set of genes involved in bacterial-host interactions, including the production of virulence factors. Conversely, the hosts can produce anti-QS compounds to impair virulence of bacterial pathogens. One of these inhibitors is the plant flavonoid naringenin, which impairs the production of QS-regulated Pseudomonas aeruginosa virulence factors. In the present work, we analyze the molecular basis for such inhibition. Our data indicate that naringenin produces its effect by directly binding the QS regulator LasR, hence competing with its physiological activator, N-(3-oxo-dodecanoyl)-L-homoserine lactone (3OC12-HSL). The in vitro analysis of LasR binding to its cognate target DNA showed that the capacity of naringenin to outcompete 3OC12-HSL, when the latter is previously bound to LasR, is low. By using an E. coli LasR-based biosensor strain, which does not produce 3OC12-HSL, we determined that the inhibition of LasR is more efficient when naringenin binds to nascent LasR than when this regulator is already activated through 3OC12-HSL binding. According to these findings, at early exponential growth phase, when the amount of 3OC12-HSL is low, naringenin should proficiently inhibit the P. aeruginosa QS response, whereas at later stages of growth, once 3OC12-HSL concentration reaches a threshold enough for binding LasR, naringenin would not efficiently inhibit the QS response. To test this hypothesis, we analyze the potential effect of naringenin over the QS response by adding naringenin to P. aeruginosa cultures at either time zero (early inhibition) or at stationary growth phase (late inhibition). In early inhibitory conditions, naringenin inhibited the expression of QS-regulated genes, as well as the production of the QS-regulated virulence factors, pyocyanin and elastase. Nevertheless, in late inhibitory conditions, the P. aeruginosa QS response was not inhibited by naringenin. Therefore, this time-dependent inhibition may compromise the efficiency of this flavonoid, which will be effective just when used against bacterial populations presenting low cellular densities, and highlight the importance of searching for QS inhibitors whose mechanism of action does not depend on the QS status of the population.

Project description:The opportunistic pathogen Pseudomonas aeruginosa is a leading cause of airway infection in cystic fibrosis (CF) patients. P. aeruginosa employs several hierarchically arranged and interconnected quorum sensing (QS) regulatory circuits to produce a battery of virulence factors such as elastase, phenazines, and rhamnolipids. The QS transcription factor LasR sits atop this hierarchy and activates the transcription of dozens of genes, including that encoding the QS regulator RhlR. Paradoxically, inactivating lasR mutations are frequently observed in isolates from CF patients with chronic P. aeruginosa infections. In contrast, mutations in rhlR are rare. We have recently shown that in CF isolates, the QS circuitry is often rewired such that RhlR acts in a LasR-independent manner. To begin understanding how QS activity differs in this rewired background, we characterized QS activation and RhlR-regulated gene expression in P. aeruginosa E90, a LasR-null, RhlR-active chronic infection isolate. In this isolate, RhlR activates the expression of 53 genes in response to increasing cell density. The genes regulated by RhlR include several that encode virulence factors. Some, but not all, of these genes are present in the QS regulon described in the well-studied laboratory strain PAO1. We also demonstrate that E90 produces virulence factors at similar concentrations as PAO1, and in E90, RhlR plays a significant role in mediating cytotoxicity in a three-dimensional lung epithelium cell model. These data illuminate a rewired LasR-independent RhlR regulon in chronic infection isolates and suggest further investigation of RhlR as a possible target for therapeutic development in chronic infections.IMPORTANCE Pseudomonas aeruginosa is a prominent cystic fibrosis (CF) pathogen that uses quorum sensing (QS) to regulate virulence. In laboratory strains, the key QS regulator is LasR. Many isolates from patients with chronic CF infections appear to use an alternate QS circuitry in which another transcriptional regulator, RhlR, mediates QS. We show that a LasR-null CF clinical isolate engages in QS through RhlR and remains capable of inducing cell death in an in vivo-like lung epithelium cell model. Our findings support the notion that LasR-null clinical isolates can engage in RhlR QS and highlight the centrality of RhlR in chronic P. aeruginosa infections.

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Dataset Information

Pseudomonas aeruginosa N-3-oxo-dodecanoyl-homoserine Lactone Elicits Changes in Cell Volume, Morphology, and AQP9 Characteristics in Macrophages.

Publications

Pseudomonas aeruginosa N-3-oxo-dodecanoyl-homoserine Lactone Elicits Changes in Cell Volume, Morphology, and AQP9 Characteristics in Macrophages.

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