CONICET | Buscador de Institutos y Recursos Humanos

Introduccion. We have recently demonstrated that in the absence of opsonic antibodies B. parapertussis (Bpp) avoids neutrophil intracellular bactericidal mechanism both by preventing bacterial uptake and by avoiding phagosome-lysosome fusion if it is phagocytosed. Objective. Investigate the relevance of non-phagocytic bactericidal mechanisms (degranulation and neutrophil extracellular traps (NETs)) of neutrophils in the control of Bpp.Materials and Methods. Isolated human neutrophils were used. Neutrophil degranulation was studied by measuring the β-glucuronidase activity in the supernatant of infected neutrophils, and by confocal microscopy using specifics markers. NET formation was studied by confocal microscopy using a DNA stain, and antibodies against NET-associated proteins.Results: Our results showed that Bpp avoids both neutrophil extracellular bactericidal mechanisms. Neutrophil degranulation preclusion seems to be dependent on the O antigen molecule that targets the bacteria to the cell lipid rafts. Bacterial entry through these domains avoids fusion of nascent phagosomes containing Bpp with specific and azhurophilic granules. IgG opsonization of Bpp impairs the bacterial ability to block neutrophil degranulation. We further observed that Bpp did not induce NETs release in resting neutrophils and had the ability to inhibit NETs formation in response to both proinflamatory and chemical NETs inducers stimuli (PMA). This latter effect proved dependent on CyaA expression showing that Bpp controls this essential neutrophil bactericidal mechanism through two different mechanisms, one related to the lack of proper NET-inducer stimuli and the other related to an active inhibition mechanism. Conclusion: Together with previous results these data suggest that Bpp subverts the main neutrophils bactericidal functions eventually precluding the efficient clearance of this bacterium in non-immune hosts.

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