Compartmentalized Cyclic AMP Production by the Bordetella pertussis and Bacillus anthracis Adenylate Cyclase Toxins Differentially Affects the Immune Synapse in T Lymphocytes

Abstract

A central feature of the immune synapse (IS) is the tight compartmentalization of membrane receptors and signaling mediators that is functional for its ability to coordinate T cell activation. Second messengers centrally implicated in this process, such as Ca2+ and diacyl glycerol, also undergo compartmentalization at the IS. Current evidence suggests a more complex scenario for cyclic AMP (cAMP), which acts both as positive and as negative regulator of T-cell antigen receptor (TCR) signaling and which, as such, must be subjected to a tight spatiotemporal control to allow for signaling at the IS. Here, we have used two bacterial adenylate cyclase toxins that produce cAMP at different subcellular localizations as the result of their distinct routes of cell invasion, namely Bordetella pertussis CyaA and Bacillus anthracis edema toxin (ET), to address the ability of the T cell to confine cAMP to the site of production and to address the impact of compartmentalized cAMP production on IS assembly and function. We show that CyaA, which produces cAMP close to the synaptic membrane, affects IS stability by modulating not only the distribution of LFA-1 and its partners talin and L-plastin, as previously partly reported but also by promoting the sustained synaptic accumulation of the A-kinase adaptor protein ezrin and protein kinase A while suppressing the β-arrestin-mediated recruitment of phosphodiesterase 4B. These effects are dependent on the catalytic activity of the toxin and can be reproduced by treatment with a non-hydrolyzable cAMP analog. Remarkably, none of these effects are elicited by ET, which produces cAMP at a perinuclear localization, despite its ability to suppress TCR signaling and T cell activation through its cAMP-elevating activity. These results show that the IS responds solely to local elevations of cAMP and provide evidence that potent compartmentalization mechanisms are operational in T cells to contain cAMP close to the site of production, even when produced at supraphysiological levels.

Distinct Spatiotemporal Distribution of Bacterial Toxin-Produced Cellular cAMP Differentially Inhibits Opsonophagocytic Signaling

“…In contrast, ET-triggered cAMP signaling partially inhibited Syk and Vav phosphorylation and had no significant effect on Pyk2 phosphorylation (Figure 2). Indeed, CyaA toxin action instantly leads to the formation of a cAMP pool in the submembranous region of the cytoplasm, which may directly modulate the signaling cascades in the local/specific target region on the cytosolic side of the membrane [50,51]. Further indirect evidence is provided by microscopy experiments where Syk [8], Pyk2 [10], and Vav [13,14] have been shown to be recruited/accumulated in the submembranous region at sites of active uptake of opsonized particles, which is the cytoplasmic subdomain where membrane proximal CyaA-mediated cAMP accumulation occurs.…”

Functional and structural consequences of epithelial cell invasion by Bordetella pertussis adenylate cyclase toxin

“…It is well known that microdomains of cAMP have pronounced effects on various signaling processes and major physiological functions in a wide variety of cells [69]. For CyaA, this has been particularly well characterized in T cells where the toxin was shown to efficiently disrupt the immunological synapse and redistribute a number of essential players in the T-cell activation cascade [70,71]. Remarkably, these effects are not elicited by another bacterial toxin, the anthrax edema factor that produces similar cytosolic levels of cAMP but originating from a perinuclear localization-where the toxin is released from late endosomes [71].…”

“…For CyaA, this has been particularly well characterized in T cells where the toxin was shown to efficiently disrupt the immunological synapse and redistribute a number of essential players in the T-cell activation cascade [70,71]. Remarkably, these effects are not elicited by another bacterial toxin, the anthrax edema factor that produces similar cytosolic levels of cAMP but originating from a perinuclear localization-where the toxin is released from late endosomes [71]. Hasan et al recently reported similar findings on human monocytes [72].…”

Functional and structural consequences of epithelial cell invasion byBordetella pertussisadenylate cyclase toxin

“…It is well known that 357 microdomains of cAMP have pronounced effects on various signaling processes and major 358 physiological functions in a wide variety of cells [64]. For CyaA, this has been particularly well 359 characterized in T cells where the toxin was shown to efficiently disrupt the immunological 360 synapse and redistribute a number of essential players in the T-cell activation cascade [65,66]. 361 Remarkably, these effects are not elicited by another bacterial toxin, the anthrax edema factor 362 that produces similar cytosolic levels of cAMP but originating from a perinuclear localization -363 where the toxin is released from late endosomes [66].…”

“…For CyaA, this has been particularly well 359 characterized in T cells where the toxin was shown to efficiently disrupt the immunological 360 synapse and redistribute a number of essential players in the T-cell activation cascade [65,66]. 361 Remarkably, these effects are not elicited by another bacterial toxin, the anthrax edema factor 362 that produces similar cytosolic levels of cAMP but originating from a perinuclear localization -363 where the toxin is released from late endosomes [66]. Hasan et al recently reported similar 364 findings on human monocytes [67].…”