Bordetella pertussis Adenylate Cyclase Toxin Blocks Induction of Bactericidal Nitric Oxide in Macrophages through cAMP-Dependent Activation of the SHP-1 Phosphatase

Abstract

The adenylate cyclase toxin–hemolysin (CyaA) plays a key role in the virulence of Bordetella pertussis. CyaA penetrates complement receptor 3–expressing phagocytes and catalyzes uncontrolled conversion of cytosolic ATP to the key second messenger molecule cAMP. This paralyzes the capacity of neutrophils and macrophages to kill bacteria by complement-dependent oxidative burst and opsonophagocytic mechanisms. We show that cAMP signaling through the protein kinase A (PKA) pathway activates Src homology domain 2 containing protein tyrosine phosphatase (SHP) 1 and suppresses production of bactericidal NO in macrophage cells. Selective activation of PKA by the cell-permeable analog N6-benzoyladenosine-3′,5′-cyclic monophosphate interfered with LPS-induced inducible NO synthase (iNOS) expression in RAW264.7 macrophages, whereas inhibition of PKA by H-89 largely restored the production of iNOS in CyaA-treated murine macrophages. CyaA/cAMP signaling induced SHP phosphatase–dependent dephosphorylation of the c-Fos subunit of the transcription factor AP-1 and thereby inhibited TLR4-triggered induction of iNOS gene expression. Selective small interfering RNA knockdown of SHP-1, but not of the SHP-2 phosphatase, rescued production of TLR-inducible NO in toxin-treated cells. Finally, inhibition of SHP phosphatase activity by NSC87877 abrogated B. pertussis survival inside murine macrophages. These results reveal that an as yet unknown cAMP-activated signaling pathway controls SHP-1 phosphatase activity and may regulate numerous receptor signaling pathways in leukocytes. Hijacking of SHP-1 by CyaA action then enables B. pertussis to evade NO-mediated killing in sentinel cells of innate immunity.

cAMP Signaling of Adenylate Cyclase Toxin Blocks the Oxidative Burst of Neutrophils through Epac-Mediated Inhibition of Phospholipase C Activity

“…4C) that the contribution of PKA activation to the inhibition of ROS production in response to fMLF could be indirect and might potentially result from inhibition of MAPK (ERK and p38) signaling toward NADPH complex assembly. This would be in line with our previous reports that activation of PKA signaling by CyaA-generated cAMP activates, by an as yet unknown mechanism, the tyrosine phosphatase SHP-1, which is known to deactivate ERK by dephosphorylation of its Thr 202 and Tyr 204 residues (13,39,54). We have measured fMLF-stimulated phosphorylation of ERK on these two sites and found it to be significantly reduced by incubation with active CyaA toxin (Fig.…”

“…4C, but it remains to be conclusively demonstrated. Intriguingly, we found previously that the CyaA/cAMP-elicited activation of SHP-1 and inhibition of ERK in monocytes was mediated by the cAMP-triggered activity of PKA and not by Epac (13,39,55). The involvement of cAMP-activated PKA signaling in promoting dephosphorylation of ERK and p38 has previously been reported in human neutrophils (56,57 [58] would actually have been Epacspecific if it was modified by a methyl group at the 29 position [63]).…”

“…The cAMP-elevating CyaA toxin enables B. pertussis to escape ROS-mediated killing Although B. pertussis bacteria are sensitive to killing by ROS, the CyaA toxin was previously shown to inhibit oxidative burst and bactericidal ROS production by neutrophils (8,13). We thus tested whether the CyaA-generated cAMP signaling alone accounts for the inhibition of oxidative burst and escape of B. pertussis to ROSmediated killing.…”

“…Previously, we found that signaling of CyaA-produced cAMP through the PKA pathway yields activation of the tyrosine phosphatase Src homology domain 2-containing protein tyrosine phosphatase 1 (SHP-1) that inhibits ERK activity in macrophages (13,39). Therefore, we assessed whether CyaA action on fMLFstimulated neutrophils interferes with the activating phosphorylation of the ERK and p38 kinases that are known to be involved in NADPH oxidase assembly.…”

“…The used strains were derived from B. pertussis Tohama I (B. pertussis cyaA-wt) that was obtained as strain CIP 81.32 from the Collection of Institute Pasteur (Paris, France). The mutant strains carrying an in-frame deletion of the cyaA open reading frame on the chromosome (B. pertussis DcyaA), or secreting an enzymatically inactive CyaA-AC 2 toxoid (B. pertussis cyaA-AC 2 ) because of insertion of a GlySer dipeptide between residues 188 and 189 of CyaA (32), were described previously (13).…”