Research Article: Ca2+ Influx and Tyrosine Kinases Trigger Bordetella Adenylate Cyclase Toxin (ACT) Endocytosis. Cell Physiology and Expression of the CD11b/CD18 Integrin Major Determinants of the Entry Route

Date Published: September 13, 2013

Publisher: Public Library of Science

Author(s): Kepa B. Uribe, César Martín, Aitor Etxebarria, David González-Bullón, Geraxane Gómez-Bilbao, Helena Ostolaza, Ludger Johannes.


Humans infected with Bordetella pertussis, the whooping cough bacterium, show evidences of impaired host defenses. This pathogenic bacterium produces a unique adenylate cyclase toxin (ACT) which enters human phagocytes and catalyzes the unregulated formation of cAMP, hampering important bactericidal functions of these immune cells that eventually cause cell death by apoptosis and/or necrosis. Additionally, ACT permeabilizes cells through pore formation in the target cell membrane. Recently, we demonstrated that ACT is internalised into macrophages together with other membrane components, such as the integrin CD11b/CD18 (CR3), its receptor in these immune cells, and GM1. The goal of this study was to determine whether ACT uptake is restricted to receptor-bearing macrophages or on the contrary may also take place into cells devoid of receptor and gain more insights on the signalling involved. Here, we show that ACT is rapidly eliminated from the cell membrane of either CR3-positive as negative cells, though through different entry routes, which depends in part, on the target cell physiology and characteristics. ACT-induced Ca2+ influx and activation of non-receptor Tyr kinases into the target cell appear to be common master denominators in the different endocytic strategies activated by this toxin. Very importantly, we show that, upon incubation with ACT, target cells are capable of repairing the cell membrane, which suggests the mounting of an anti-toxin cell repair-response, very likely involving the toxin elimination from the cell surface.

Partial Text

Adenylate cyclase toxin (ACT), a ≈ 200 kDa protein, is an essential virulence factor secreted by Bordetella pertussis[1], the whooping cough bacterium. Whooping cough or pertussis is a severe infant respiratory disease that is deadly again in spite of the high vaccine coverage, due to the worldwide resurgence of more virulent forms of the bacterium [2]. ACT is a member of the calcium-binding, pore-forming RTX (Repeats-in-Toxin) family of protein toxins that share a characteristic calcium-binding motif of Gly- and Asp-rich nonapeptide repeats and a marked cytotoxic and cytolytic activity [3]–[5]. ACT exhibits besides a distinctive feature: it has an amino-terminal adenylate cyclase catalytic domain (AC domain). The RTX component forms pores, apparently oligomeric, in cell membranes and serves as the cell binding domain [6], [7]. The AC domain converts ATP into cAMP in a reaction that is stimulated by eukaryotic calmodulin [8]. To generate cAMP, ACT binds to cells, translocates the AC domain directly across the plasma membrane [9] and catalyzes the unregulated conversion of intracellular ATP to cAMP.

We present here a detailed study addressing the endocytosis of ACT in four different cell types, CHO-K1 cells, CR3+CHO-K1 cells, in which the CR3 has been stably transfected, J774A.1 cells naturally expressing CR3 and CR3−J774A.1 cells, macrophages in which expression of the CD11b chain has been knocked down. And we show that ACT is rapidly eliminated from the cell membrane either in CR3-positive as negative cells, though through different routes of entry depending on the cell type. Two key factors, the activation of non-receptor Tyrosine kinases, and the elevation of the intracellular calcium, both induced by ACT action, are involved in the ACT endocytosis pathways.