Adenylate Cyclase Toxin promotes bacterial internalisation into non phagocytic cells

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