Pharmacological Activation of Rap1 Antagonizes the Endothelial Barrier Disruption Induced by Exotoxins ExoS and ExoT of Pseudomonas aeruginosa

Abstract

Most clinical strains of Pseudomonas aeruginosa, a leading agent of nosocomial infections, are multiresistant to antibiotherapy. Because of the paucity of new available antibiotics, the investigation of strategies aimed at limiting the action of its major virulence factors has gained much interest. The type 3 secretion system of P. aeruginosa and its effectors are known to be major determinants of toxicity and are required for bacterial dissemination in the host. Bacterial transmigration across the vascular wall is considered to be an important step in the infectious process. Using human endothelial primary cells, we demonstrate that forskolin (FSK), a drug inducing cyclic AMP (cAMP) elevation in eukaryotic cells, strikingly reduced the cell retraction provoked by two type 3 toxins, ExoS and ExoT, found in the majority of clinical strains. Conversely, cytotoxicity of a strain carrying the type 3 effector ExoU was unaffected by FSK. In addition, FSK altered the capacity of two ExoS/ExoT strains to transmigrate across cell monolayers. In agreement with these findings, other drugs and a cytokine inducing the increase of cAMP intracellular levels have also protected cells from retraction. cAMP is an activator of both protein kinase A and EPAC, a GTPase exchange factor of Rap1. Using activators or inhibitors of either pathway, we show that the beneficial effect of FSK is exerted by the activation of the EPAC/Rap1 axis, suggesting that its protective effect is mediated by reinforcing cell-cell and cell-substrate adhesion. Pseudomonas aeruginosa is an opportunistic pathogen and a leading agent of nosocomial infections. The largest cohorts of P. aeruginosa-infected patients are found in three pathological settings: ventilator-associated pneumonia, bacteremia, and cystic fibrosis. In acute infections, P. aeruginosa disseminates from the primary infection site to the blood and other organs, leading to sepsis and multiple organ failure. From a clinical point of view, vascular barrier breakdown is thus considered to be a key step in the pathophysiology of infection (1).Most P. aeruginosa clinical isolates are multidrug or even extremely drug resistant to antibiotics, which explains the high fatality rates of P. aeruginosa infections. The pathogen has been recently included in a family of so-called "ESKAPE" bacterial pathogens, a group which also includes Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, and Enterobacter species, that are able to efficiently "escape" the effects of available antibacterial drugs and for which there is an urgent need for developments of novel types of drugs (2, 3). In this context, the investigation of new strategies limiting the action of the virulence factors rather than bactericidal agents has gained much interest.P. aeruginosa is extremely well equipped in virulence determinants, which are membrane-embedded protein machineries dedicated for effector/toxin export (4). The type 3 secretion system (T3SS) and its effectors are recognized as the most i...

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“…A fixed concentration of CT was mixed with different concentrations of GM1-NPs or PEG-NPs, and the mixtures were added to monolayers of human HCA7 intestinal epithelial cells. As a functional read-out for CT bioactivity, we determined levels of secreted cAMP in the supernatants, which correlate closely with intracellular cAMP levels [ 25 ]. GM1-NPs neutralized the ability of CT to activate cAMP production and secretion in a concentration-dependent fashion, while the GM1-free control PEG-NPs had no effect ( Fig 3A ).…”

Pseudomonas aeruginosa Takes a Multi-Target Approach to Achieve Junction Breach

“…The inhibition of Rho and Rac by ExoS/ExoT may thus directly affect the organization of the AJ complex. Interestingly, the pharmacological activation of another GTPase, Rap1, which also drives AJ complex organization, was shown to counteract the effect of ExoS/ExoT on junction disruption and to reduce exotoxin-induced cell rounding (Bouillot et al, 2015 ). In addition, ExoS has been shown to use unexplored mechanisms to displace ZO-1 and occludin from the TJ (Soong et al, 2008 ).…”

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“…4a ). Among molecules targeting the eukaryotic cells, the prostaglandin PGE2 and forskolin, a cAMP inducer recently shown to inhibit P. aeruginosa T3SS effects through Rap1 activation 12 , significantly delayed the kinetics (Fig. 4c ).…”

“…Furthermore, the screening approach was successfully applied to a set of antibodies and small molecules targeting either bacteria or eukaryotic cells and to a panel of siRNAs. Indeed, it identified three inhibitory activities among the tested small molecules and antibodies: the MBX2401 drug and anti-PcrV antibodies, both known to inhibit P. aeruginosa T3SS 16 , 17 , and forskolin, known to counteract the T3SS effect in eukaryotic cells 12 . This represents a proof of concept for a screening strategy.…”