calpain and Pseudomonas-Infections

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 Ca(2+) activity as well as Ca(2+) -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.

Topics: Adult; Aged; Aged, 80 and over; Blood Coagulation; Calcium; Calpain; Cations, Divalent; Ceramides; Eryptosis; Erythrocytes; Female; Fibrin; Humans; Ion Transport; Male; Middle Aged; Phosphatidylserines; Prothrombin; Pseudomonas aeruginosa; Pseudomonas Infections; Pyocyanine; Reactive Oxygen Species; Sepsis; Virulence Factors

Cystic fibrosis transmembrane conductance regulator (CFTR) protein is a chloride channel regulating fluid homeostasis at epithelial surfaces. Its loss of function induces hypohydration, mucus accumulation, and bacterial infections in CF and potentially other lung chronic diseases.. To test whether neutrophil elastase (NE) and neutrophil-mediated inflammation negatively impact CFTR structure and function, in vitro and in vivo.. Using an adenovirus-CFTR overexpression approach, we showed that NE degrades wild-type (WT)- and ΔF508-CFTR in vitro and WT-CFTR in mice through a new pathway involving the activation of intracellular calpains.. CFTR degradation triggered a loss of function, as measured in vitro by channel patch-clamp and in vivo by nasal potential recording in mice. Importantly, this mechanism was also shown to be operative in a Pseudomonas aeruginosa lung infection murine model, and was NE-dependent, because CFTR integrity was significantly protected in NE(-/-) mice compared with WT mice.. These data provide a new mechanism and show for the first time a link between NE-calpains activation and CFTR loss of function in bacterial lung infections relevant to CF and to other chronic inflammatory lung conditions.

Topics: Animals; Calpain; Chloride Channels; Cystic Fibrosis Transmembrane Conductance Regulator; Epithelium; Humans; Leukocyte Elastase; Lung; Male; Mice; Mice, Inbred C57BL; Patch-Clamp Techniques; Pneumonia, Bacterial; Pseudomonas Infections

Recruitment of polymorphonuclear leukocytes (PMNs) into the lungs in response to inhaled pathogens is initiated by epithelial signaling, the activation of toll-like receptors (TLRs), and the production of the chemokine interleukin-8. To reach the site of infection, PMNs must be mobilized through epithelial junctions. Here, we demonstrate that Ca(2+) fluxes generated by TLR2 signals activate calpains, Ca(2+)-dependent cysteine proteases. These activated calpains cleave the transmembrane junctional proteins occludin and E-cadherin without breaching the integrity of the epithelial barrier. Calpain inhibitors decrease PMN transepithelial migration in response to TLR2 agonists both in vitro and in a mouse model of P. aeruginosa infection. Thus, TLR2 signaling in the airway not only induces chemokine expression to recruit PMNs, but also initiates cleavage of junctional proteins to accommodate transmigration of the recruited PMNs.

Topics: Animals; Cadherins; Calcium; Calpain; Cell Line; Cell Migration Assays, Leukocyte; Cell Movement; Epithelial Cells; Humans; Intercellular Junctions; Leukocytes; Membrane Proteins; Mice; Mice, Inbred C57BL; Occludin; Pneumonia, Bacterial; Pseudomonas Infections; Toll-Like Receptor 2