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Bordetella pertussis causes whooping cough, a respiratory infectious disease that is the fifth largest cause of vaccine-preventable death in infants. Though historically considered an extracellular pathogen, this bacterium has been detected both in vitro and in vivo inside phagocytic and non-phagocytic cells. However the precise mechanism used by B. pertussis for cell entry, or the putative bacterial factors involved, are not fully elucidated. Here we find that adenylate cyclase toxin (ACT), one of the important toxins of B. pertussis, is sufficient to promote bacterial internalisation into non-phagocytic cells. After characterization of the entry route we show that uptake of "toxin-coated bacteria" proceeds via a clathrin-independent, caveolae-dependent entry pathway, allowing the internalised bacteria to survive within the cells. Intracellular bacteria were found inside non-acidic endosomes with high sphingomyelin and cholesterol content, or "free" in the cytosol of the invaded cells, suggesting that the ACT-induced bacterial uptake may not proceed through formation of late endolysosomes. Activation of Tyr kinases and toxin-induced Ca2+-influx are essential for the entry process. We hypothesize that B. pertussis might use ACT to activate the endocytic machinery of non-phagocytic cells and gain entry into these cells, in this way evading the host immune system. Technical and human support provided by SGIker (Analytical and High-Resolution Microscopy in Biomedicine Service of UPV/EHU) and Rocio Alonso for excellent technical assistance are gratefully acknowledged. We also thank Dr. N. Guiso from the Pasteur Institute (Paris, France) for the Bordetella pertussis strains BP18323 and non-virulent bgv negative BP18323H-. This study was supported by grants from the Spanish Ministerio de Ciencia y Tecnologia (Project BFU 2012-36241), and the Basque Government (Grupos Consolidados IT849-13 and ETORTEK Program). K.B.U and D.G.B. were recipients of a fellowship from the Bizkaia Biophysics Foundation. A.E.G. was a recipient of a research contract (INNPACTO 2010) and A.E.L. was a recipient of a fellowship from the University of the Basque Country (UPV/EH). A patent with reference N/Ref.: P1683USPC (2004) was approved.

Perforin-mediated cytotoxicity is an essential host defense, in which defects contribute to tumor development and pathogenic disorders including autoimmunity and autoinflammation. How perforin (PFN) facilitates intracellular delivery of pro-apoptotic and inflammatory granzymes across the bilayer of targets remains unresolved. Here we show that cellular susceptibility to granzyme B (GzmB) correlates with rapid PFN-induced phosphatidylserine externalization, suggesting that pores are formed at a protein-lipid interface by incomplete membrane oligomers (or arcs). Supporting a role for these oligomers in protease delivery, an anti-PFN antibody (pf-80) suppresses necrosis but increases phosphatidylserine flip-flop and GzmB-induced apoptosis. As shown by atomic force microscopy on planar bilayers and deep-etch electron microscopy on mammalian cells, pf-80 increases the proportion of arcs which correlates with the presence of smaller electrical conductances, while large cylindrical pores decline. PFN appears to form arc structures on target membranes that serve as minimally disrupting conduits for GzmB translocation. The role of these arcs in PFN-mediated pathology warrants evaluation where they may serve as novel therapeutic targets.Cell Death and Differentiation advance online publication, 22 August 2014; doi:10.1038/cdd.2014.110.