RESUMO
Panton-Valentine Leukocidin (PVL) is a bicomponent leukotoxin produced by 3%-10% of clinical Staphylococcus aureus (SA) strains involved in the severity of hospital and community-acquired infections. Although PVL was long known as a pore-forming toxin, recent studies have challenged the formation of a pore at the plasma membrane, while its endocytosis and the exact mode of action remain to be defined. In vitro immunolabeling of human neutrophils shows that Neutrophil Extracellular Traps (NETosis) is triggered by the action of purified PVL, but not by Gamma hemolysin CB (HlgCB), a structurally similar SA leukotoxin. PVL causes the ejection of chromatin fibers (NETs) decorated with antibacterial peptides independently of the NADPH oxidase oxidative burst. Leukotoxin partially colocalizes with mitochondria and enhances the production of reactive oxygen species from these organelles, while showing an increased autophagy, which results unnecessary for NETs ejection. PVL NETosis is elicited through Ca2+ -activated SK channels and Myeloperoxidase activity but is abolished by Allopurinol pretreatment of neutrophils. Moreover, massive citrullination of the histone H3 is performed by peptidyl arginine deiminases. Inhibition of this latter enzymes fails to abolish NET extrusion. Unexpectedly, PVL NETosis does not seem to involve Src kinases, which is the main kinase family activated downstream the binding of PVL F subunit to CD45 receptor, while the specific kinase pathway differs from the NADPH oxidase-dependent NETosis. PVL alone causes a different and specific form of NETosis that may rather represent a bacterial strategy conceived to disarm and disrupt the immune response, eventually allowing SA to spread.
Assuntos
Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/metabolismo , Exotoxinas/metabolismo , Armadilhas Extracelulares/imunologia , Leucocidinas/metabolismo , Mitocôndrias/metabolismo , Neutrófilos/imunologia , Infecções Estafilocócicas/imunologia , Staphylococcus aureus/metabolismo , Adulto , Células Cultivadas , Feminino , Voluntários Saudáveis , Proteínas Hemolisinas/metabolismo , Humanos , Masculino , NADPH Oxidases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Explosão Respiratória , Infecções Estafilocócicas/microbiologiaRESUMO
A growing number of receptors, often associated with the innate immune response, are being identified as targets for bacterial toxins of the beta-stranded pore-forming family. These findings raise the new question of whether the receptors are activated or merely used as docking points facilitating the formation of a pore. To elucidate whether the Staphylococcus aureusâ Panton-Valentine leukocidin and the leukotoxin HlgC/HlgB act through the C5a receptor (C5aR) as agonists, antagonists or differ from the C5a complement-derived peptide, their activity is explored on C5aR-expressing cells. Both leukotoxins equally bound C5aR in neutrophils and in stable transfected U937 cells and initiated mobilization of intracellular Ca(2+) . HlgC/HlgB requires the presence of robust intracellular acidic Ca(2+) stores in order to evoke a rise in free [Ca(2+) ]i , while the LukS-PV/LukF-PV directly altered reticular Ca(2+) stores. Intracellular target specificity is conferred by the F-subunit associated to the S-subunit binding the receptor. Furthermore, internalization of the two leukotoxin components (S- and F-subunits) associated to C5aR is required for the initiation of [Ca(2+) ]i mobilization. Electrophysiological recordings on living cells demonstrated that LukS-PV/LukF-PV does not alter the membrane resistance of C5aR-expressing cells. The present observations suggest that part of the pore-forming process occurs in distinct intracellular compartments rather than at the plasma membrane.