Jiri Masin

Inflammasome Activation by Adenylate Cyclase Toxin Directs Th17 Responses and Protection against Bordetella pertussis

Inflammasome-mediated IL-1β production is central to the innate immune defects that give rise to certain autoinflammatory diseases and may also be associated with the generation of IL-17–producing CD4+ T (Th17) cells that mediate autoimmunity. However, the role of the inflammasome in driving adaptive immunity to infection has not been addressed. In this article, we demonstrate that inflammasome-mediated IL-1β plays a critical role in promoting Ag-specific Th17 cells and in generating protective immunity against Bordetella pertussis infection. Using a murine respiratory challenge model, we demonstrated that the course of B. pertussis infection was significantly exacerbated in IL-1R type I-defective (IL-1RI−/−) mice. We found that adenylate cyclase toxin (CyaA), a key virulence factor secreted by B. pertussis, induced robust IL-1β production by dendritic cells through activation of caspase-1 and the NALP3-containing inflammasome complex. Using mutant toxins, we demonstrate that CyaA-mediated activation of caspase-1 was not dependent on adenylate cyclase enzyme activity but was dependent on the pore-forming capacity of CyaA. In addition, CyaA promoted the induction of Ag-specific Th17 cells in wild-type but not IL-1RI−/− mice. Furthermore, the bacterial load was enhanced in IL-17–defective mice. Our findings demonstrate that CyaA, a virulence factor from B. pertussis, promotes innate IL-1β production via activation of the NALP3 inflammasome and, thereby, polarizes T cell responses toward the Th17 subtype. In addition to its known role in subverting host immunity, our findings suggest that CyaA can promote IL-1β–mediated Th17 cells, which promote clearance of the bacteria from the respiratory tract.

Adenylate Cyclase Toxin Subverts Phagocyte Function by RhoA Inhibition and Unproductive Ruffling

Adenylate cyclase toxin (CyaA or ACT) is a key virulence factor of pathogenic Bordetellae. It penetrates phagocytes expressing the αMβ2 integrin (CD11b/CD18, Mac-1 or CR3) and paralyzes their bactericidal capacities by uncontrolled conversion of ATP into a key signaling molecule, cAMP. Using pull-down activity assays and transfections with mutant Rho family GTPases, we show that cAMP signaling of CyaA causes transient and selective inactivation of RhoA in mouse macrophages in the absence of detectable activation of Rac1, Rac2, or RhoG. This CyaA/cAMP-induced drop of RhoA activity yielded dephosphorylation of the actin filament severing protein cofilin and massive actin cytoskeleton rearrangements, which were paralleled by rapidly manifested macrophage ruffling and a rapid and unexpected loss of macropinocytic fluid phase uptake. As shown in this study for the first time, CyaA/cAMP signaling further caused a rapid and near-complete block of complement-mediated phagocytosis. Induction of unproductive membrane ruffling, hence, represents a novel sophisticated mechanism of down-modulation of bactericidal activities of macrophages and a new paradigm for action of bacterial toxins that hijack host cell signaling by manipulating cellular cAMP levels.

RTX cytotoxins recognize β2 integrin receptors through N-linked oligosaccharides

Bordetella pertussis adenylate cyclase (AC) toxin–hemolysin (Hly) (CyaA, ACT, or AC-Hly) is a cytotoxin of the RTX (repeat in toxin) family. It delivers into target cells an AC domain that catalyzes uncontrolled conversion of ATP to cAMP, a key signaling molecule subverting phagocyte functions. CyaA utilizes a heavily N-glycosylated β2 integrin receptor CD11b/CD18 (αMβ2, Mac-1, or CR3). We show that deglycosylation of cell surface proteins by glycosidase treatment, or inhibition of protein N-glycosylation by tunicamycin, ablates CyaA binding and penetration of CD11b-expressing cells. Furthermore, binding of CyaA to cells was strongly inhibited in the presence of free saccharides occurring as building units of integrin oligosaccharide complex, whereas saccharides absent from integrin oligosaccharide chains failed to inhibit CyaA binding to CD11b/CD18-expressing cells. CyaA, hence, selectively recognized sugar residues of N-linked oligosaccharides of integrins. Moreover, glycosylation of CD11a/CD18, another receptor of the β2 integrin family, was also essential for cytotoxic action of other RTX cytotoxins, the leukotoxin of Aggregatibacter actinomycetemcomitans (LtxA) and the Escherichia coli α-Hly (HlyA). These results show that binding and killing of target cells by CyaA, LtxA, and HlyA depends on recognition of N-linked oligosaccharide chains of β2 integrin receptors. This sets a new paradigm for action of RTX cytotoxins.

Pore-Forming and Enzymatic Activities of Bordetella pertussis Adenylate Cyclase Toxin Synergize in Promoting Lysis of Monocytes

ABSTRACT Bordetella adenylate cyclase (AC) toxin-hemolysin (CyaA) targets myeloid phagocytes expressing the αMβ2 integrin (CD11b/CD18) and delivers into their cytosol an AC enzyme that converts ATP into cyclic AMP (cAMP). In parallel, CyaA acts as a hemolysin, forming small membrane pores. Using specific mutations, we dissected the contributions of the two activities to cytolytic potency of CyaA on J774A.1 murine monocytes. The capacity of AC to penetrate cells and deplete cytosolic ATP was essential for promoting lysis and the enzymatically inactive but fully hemolytic CyaA-AC− toxoid exhibited a 15-fold-lower cytolytic capacity on J774A.1 cells than intact CyaA. Moreover, a two- or fourfold drop of specific hemolytic activity of the CyaA-E570Q and CyaA-E581P mutants was overpowered by an intact capacity to dissipate cytosolic ATP into cAMP, allowing the less hemolytic proteins to promote lysis of J774A.1 cells as efficiently as intact CyaA. However, an increased hemolytic activity, due to lysine substitutions of glutamates 509, 516, and 581 in the pore-forming domain, conferred on AC− toxoids a correspondingly enhanced cytolytic potency. Moreover, a threefold increase in hemolytic activity could override a fourfold drop in capacity to convert cellular ATP to cAMP, conferring on the CyaA-E581K construct an overall twofold increased cytolytic potency. Hence, although appearing auxiliary in cytolytic action of the toxin on nucleated cells, the pore-forming activity can synergize with ATP-depleting activity of the cell-invasive AC enzyme and complement its action toward maximal cytotoxicity.

Bordetella Adenylate Cyclase Toxin Mobilizes Its β2 Integrin Receptor into Lipid Rafts to Accomplish Translocation across Target Cell Membrane in Two Steps

Bordetella adenylate cyclase toxin (CyaA) binds the αMβ2 integrin (CD11b/CD18, Mac-1, or CR3) of myeloid phagocytes and delivers into their cytosol an adenylate cyclase (AC) enzyme that converts ATP into the key signaling molecule cAMP. We show that penetration of the AC domain across cell membrane proceeds in two steps. It starts by membrane insertion of a toxin ‘translocation intermediate’, which can be ‘locked’ in the membrane by the 3D1 antibody blocking AC domain translocation. Insertion of the ‘intermediate’ permeabilizes cells for influx of extracellular calcium ions and thus activates calpain-mediated cleavage of the talin tether. Recruitment of the integrin-CyaA complex into lipid rafts follows and the cholesterol-rich lipid environment promotes translocation of the AC domain across cell membrane. AC translocation into cells was inhibited upon raft disruption by cholesterol depletion, or when CyaA mobilization into rafts was blocked by inhibition of talin processing. Furthermore, CyaA mutants unable to mobilize calcium into cells failed to relocate into lipid rafts, and failed to translocate the AC domain across cell membrane, unless rescued by Ca2+ influx promoted in trans by ionomycin or another CyaA protein. Hence, by mobilizing calcium ions into phagocytes, the ‘translocation intermediate’ promotes toxin piggybacking on integrin into lipid rafts and enables AC enzyme delivery into host cytosol.

Third Activity of Bordetella Adenylate Cyclase (AC) Toxin-Hemolysin MEMBRANE TRANSLOCATION OF AC DOMAIN POLYPEPTIDE PROMOTES CALCIUM INFLUX INTO CD11b+ MONOCYTES INDEPENDENTLY OF THE CATALYTIC AND HEMOLYTIC ACTIVITIES

The Bordetella adenylate cyclase toxin-hemolysin (CyaA) targets phagocytes expressing the αMβ2 integrin (CD11b/CD18), permeabilizes their membranes by forming small cation-selective pores, and delivers into cells a calmodulin-activated adenylate cyclase (AC) enzyme that dissipates cytosolic ATP into cAMP. We describe here a third activity of CyaA that yields elevation of cytosolic calcium concentration ([Ca2+]i) in target cells. The CyaA-mediated [Ca2+]i increase in CD11b+ J774A.1 monocytes was inhibited by extracellular La3+ ions but not by nifedipine, SK&F 96365, flunarizine, 2-aminoethyl diphenylborinate, or thapsigargin, suggesting that influx of Ca2+ into cells was not because of receptor signaling or opening of conventional calcium channels by cAMP. Compared with intact CyaA, a CyaA-AC– toxoid unable to generate cAMP promoted a faster, albeit transient, elevation of [Ca2+]i. This was not because of cell permeabilization by the CyaA hemolysin pores, because a mutant exhibiting a strongly enhanced pore-forming activity (CyaA-E509K/E516K), but unable to deliver the AC domain into cells, was also unable to elicit a [Ca2+]i increase. Further mutations interfering with AC translocation into cells, such as proline substitutions of glutamate residues 509 or 570 or deletion of the AC domain as such, reduced or ablated the [Ca2+]i-elevating capacity of CyaA. Moreover, structural alterations within the AC domain, because of insertion of various oligopeptides, differently modulated the kinetics and extent of Ca2+ influx elicited by the respective AC– toxoids. Hence, the translocating AC polypeptide itself appears to participate in formation of a novel type of membrane path for calcium ions, contributing to action of CyaA in an unexpected manner.

Segments Crucial for Membrane Translocation and Pore-forming Activity of Bordetella Adenylate Cyclase Toxin

Bordetella adenylate cyclase toxin-hemolysin (CyaA, AC-Hly, or ACT) permeabilizes cell membranes by forming small cation-selective (hemolytic) pores and subverts cellular signaling by delivering into host cells an adenylate cyclase (AC) enzyme that converts ATP to cAMP. Both AC delivery and pore formation were previously shown to involve a predicted amphipathic α-helix502–522 containing a pair of negatively charged Glu509 and Glu516 residues. Another predicted transmembrane α-helix565–591 comprises a Glu570 and Glu581 pair. We examined the roles of these glutamates in the activity of CyaA. Substitutions of Glu516 increased specific hemolytic activity of CyaA by two different molecular mechanisms. Replacement of Glu516 by positively charged lysine residue (E516K) increased the propensity of CyaA to form pores, whereas proline (E516P) or glutamine (E516Q) substitutions extended the lifetime of open single pore units. All three substitutions also caused a drop of pore selectivity for cations. Substitutions of Glu570 and Glu581 by helix-breaking proline or positively charged lysine residue reduced (E570K, E581P) or ablated (E570P, E581K) AC membrane translocation. Moreover, E570P, E570K, and E581P substitutions down-modulated also the specific hemolytic activity of CyaA. In contrast, the E581K substitution enhanced the hemolytic activity of CyaA 4 times, increasing both the frequency of formation and lifetime of toxin pores. Negative charge at position 570, but not at position 581, was found to be essential for cation selectivity of the pore, suggesting a role of Glu570 in ion filtering inside or close to pore mouth. The pairs of glutamate residues in the predicted transmembrane segments of CyaA thus appear to play a key functional role in membrane translocation and pore-forming activities of CyaA.

Membrane Restructuring by Bordetella pertussis Adenylate Cyclase Toxin, a Member of the RTX Toxin Family

Adenylate cyclase toxin (ACT) is secreted by Bordetella pertussis, the bacterium causing whooping cough. ACT is a member of the RTX (repeats in toxin) family of toxins, and like other members in the family, it may bind cell membranes and cause disruption of the permeability barrier, leading to efflux of cell contents. The present paper summarizes studies performed on cell and model membranes with the aim of understanding the mechanism of toxin insertion and membrane restructuring leading to release of contents. ACT does not necessarily require a protein receptor to bind the membrane bilayer, and this may explain its broad range of host cell types. In fact, red blood cells and liposomes (large unilamellar vesicles) display similar sensitivities to ACT. A varying liposomal bilayer composition leads to significant changes in ACT-induced membrane lysis, measured as efflux of fluorescent vesicle contents. Phosphatidylethanolamine (PE), a lipid that favors formation of nonlamellar (inverted hexagonal) phases, stimulated ACT-promoted efflux. Conversely, lysophosphatidylcholine, a micelle-forming lipid that opposes the formation of inverted nonlamellar phases, inhibited ACT-induced efflux in a dose-dependent manner and neutralized the stimulatory effect of PE. These results strongly suggest that ACT-induced efflux is mediated by transient inverted nonlamellar lipid structures. Cholesterol, a lipid that favors inverted nonlamellar phase formation and also increases the static order of phospholipid hydrocarbon chains, among other effects, also enhanced ACT-induced liposomal efflux. Moreover, the use of a recently developed fluorescence assay technique allowed the detection of trans-bilayer (flip-flop) lipid motion simultaneous with efflux. Lipid flip-flop further confirms the formation of transient nonlamellar lipid structures as a result of ACT insertion in bilayers.

Prime/boost immunotherapy of HPV16-induced tumors with E7 protein delivered by Bordetella adenylate cyclase and modified vaccinia virus Ankara

The Bordetella adenylate cyclase toxoid (CyaA) targets cells expressing the αMβ2 integrin receptor CD11b/CD18 (CR3 or Mac-1) and can penetrate into cytosol of professional antigen-presenting cells, such as dendritic cells. This allows us to use CyaA for delivery of passenger antigens into the cytosolic pathway of processing and MHC class I-restricted presentation, which can promote induction of antigen-specific CD8+ cytotoxic T-lymphocyte immune responses. We show here that vaccination with a genetically detoxified CyaA336/E7 protein, carrying the full-length oncoprotein E7 of the human papilloma virus 16 inserted at position 336 of the cell-invasive AC domain of CyaA, induces an E7-specific CD8+ T-cell immune response and confers on mice protective, as well as therapeutic immunity against challenge with TC-1 tumor cells expressing the E7 oncoprotein. The therapeutic efficacy of priming with the CyaA336/E7 vaccine could further be enhanced by a heterologous booster immunization with a highly attenuated modified vaccinia virus Ankara (MVA) expressing the E7 protein fused to the lysosome-associated membrane protein (LAMP1). These results establish the potential of CyaA as a new antigen delivery tool for prime/boost immunotherapy of tumors.

Adenylate cyclase toxin translocates across target cell membrane without forming a pore

The adenylate cyclase toxin‐haemolysin of Bordetella (CyaA) targets CD11b+ myeloid phagocytes and translocates across their cytoplasmic membrane an adenylate cyclase (AC) enzyme that catalyses conversion of cytosolic ATP into cAMP. In parallel, CyaA acts as a cytolysin forming cation‐selective pores, which permeabilize cell membrane and eventually provoke cell lysis. Using cytolytic activity, potassium efflux and patch‐clamp assays, we show that a combination of substitutions within the pore‐forming (E570Q) and acylation‐bearing domain (K860R) ablates selectively the cell‐permeabilizing activity of CyaA. At the same time, however, the capacity of such mutant CyaA to translocate the AC domain across cytoplasmic membrane into cytosol of macrophage cells and to elevate cellular cAMP concentrations remained intact. Moreover, the combination of E570Q+K860R substitutions suppressed the residual cytolytic activity of the enzymatically inactive CyaA/OVA/AC‐ toxoid on CD11b‐expressing monocytes, while leaving unaffected the capacity of the mutant toxoid to deliver in vitro a reporter CD8+ T cell epitope from ovalbumin (OVA) to the cytosolic pathway of dendritic cells for MHC class I‐restricted presentation and induce in vivo an OVA‐specific cytotoxic T cell response. CyaA, hence, employs a mechanism of AC enzyme domain translocation across cellular membrane that avoids passage across the cytolytic pore formed by toxin oligomers.