Abstract
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.
Highlights
Reports had suggested that adenylate cyclase toxin (ACT) was not involved in invasion, as mutant B. pertussis strains lacking ACT were capable of invading HeLa 229 cells, others have suggested that ACT inhibits bacterial invasion in human tracheal epithelial cells (HTE) and in HeLa cells[6,7] and other group did not found evidence for a significant inhibitory effect of ACT in the entry of B. pertussis into A549 cells[10]
Bacterial uptake is normally preceded by perturbations of the cellular cytoskeleton, as documented for the invasive pathogenic species Listeria monocytogenes and Salmonella typhimurium[35]
The α Mβ 2 integrin is a bona fide phagocytic receptor for professional phagocytes with a central role in microbial uptake[36,37,38]; ligand binding to the integrin may activate phagocytosis[38]
Summary
Our group has reported that purified ACT is internalised by both phagocytic (J774A.1 macrophages) and non-phagocytic cells (CHO-K1) through activation of different entry pathways depending on the cell type[32]. We sought to determine whether the ACT molecules surrounding the bacteria might be able to induce the internalisation of B. pertussis into non-phagocytic cells. For this purpose, we employed two bacterial strains, B. pertussis strain BP18323 which expresses the bgv-regulated virulence factors including ACT34, and a non-virulent strain that lacks the bgv determinant, and cannot express the bgv-regulated virulence factors (strain BP18323H-)[34]. We show here that purified ACT is sufficient to promote internalisation of live bacteria into non-phagocytic CHO-K1 cells, and that internalised bacteria survive within the cells
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