Radim Osicka

RTX proteins: a highly diverse family secreted by a common mechanism

Repeats-in-toxin (RTX) exoproteins of Gram-negative bacteria form a steadily growing family of proteins with diverse biological functions. Their common feature is the unique mode of export across the bacterial envelope via the type I secretion system and the characteristic, typically nonapeptide, glycine- and aspartate-rich repeats binding Ca2+ ions. In this review, we summarize the current state of knowledge on the organization of rtx loci and on the biological and biochemical activities of therein encoded proteins. Applying several types of bioinformatic screens on the steadily growing set of sequenced bacterial genomes, over 1000 RTX family members were detected, with the biological functions of most of them remaining to be characterized. Activities of the so far characterized RTX family members are then discussed and classified according to functional categories, ranging from the historically first characterized pore-forming RTX leukotoxins, through the large multifunctional enzymatic toxins, bacteriocins, nodulation proteins, surface layer proteins, up to secreted hydrolytic enzymes exhibiting metalloprotease or lipase activities of industrial interest.

Interaction of Bordetella pertussis adenylate cyclase with CD11b/CD18: Role of toxin acylation and identification of the main integrin interaction domain

Adenylate cyclase toxin (CyaA) is one of the major virulence factors produced by Bordetella pertussis, the whooping cough agent. CyaA belongs to the repeat in toxin protein family and requires a post-translational fatty acylation to form cation-selective channels in target cell membranes and to penetrate into cytosol. We have demonstrated recently that CyaA uses the αMβ2 integrin (CD11b/CD18) as a specific cellular receptor. Here we show that the acylation of CyaA is required for a productive and tight interaction of the toxin with cells expressing CD11b. In addition, we demonstrate that the catalytic domain is not required for binding of CyaA to CD11b and that the main integrin interacting domain of CyaA is located in its glycine/aspartate-rich repeat region. These data decipher, for the first time, the interaction of CyaA with CD11b-positive cells and open new prospects for understanding the interaction of Bordetella pertussis with innate and adaptive immune systems.

Integrin subunit CD18 Is the T-lymphocyte receptor for the Helicobacter pylori vacuolating cytotoxin

Helicobacter pylori infection is associated with gastritis, ulcerations, and gastric adenocarcinoma. H. pylori secretes the vacuolating cytotoxin (VacA), a major pathogenicity factor. VacA has immunosuppressive effects, inhibiting interleukin-2 (IL-2) secretion by interference with the T cell receptor/IL-2 signaling pathway at the level of calcineurin, the Ca2+-calmodulin-dependent phosphatase. Here, we show that VacA efficiently enters activated, migrating primary human T lymphocytes by binding to the beta2 (CD18) integrin receptor subunit and exploiting the recycling of lymphocyte function-associated antigen (LFA)-1. LFA-1-deficient Jurkat T cells were resistant to vacuolation and IL-2 modulation, and genetic complementation restored sensitivity to VacA. VacA targeted human, but not murine, CD18 for cell entry, consistent with the species-specific adaptation of H. pylori. Furthermore, expression of human integrin receptors (LFA-1 or Mac-1) in murine T cells resulted in VacA-mediated cellular vacuolation. Thus, H. pylori co-opts CD18 as a VacA receptor on human T lymphocytes to subvert the host immune response.

Delivery of CD8+ T-Cell Epitopes into Major Histocompatibility Complex Class I Antigen Presentation Pathway by Bordetella pertussis Adenylate Cyclase: Delineation of Cell Invasive Structures and Permissive Insertion Sites

ABSTRACT Bordetella pertussis adenylate cyclase (AC) toxin-hemolysin (ACT-Hly) can penetrate a variety of eukaryotic cells. Recombinant AC toxoids have therefore been recently used for delivery of CD8+ T-cell epitopes into antigen-presenting cells in vivo and for induction of protective antiviral, as well as therapeutic antitumor cytotoxic T-cell responses. We have explored the carrier potential of the ACT molecule by insertional mutagenesis scanning for new permissive sites, at which integration of two- to nine-residue-long peptides does not interfere with membrane interaction and translocation of ACT. A model CD8+ T-cell epitope of ovalbumin was incorporated at 10 of these permissive sites along the toxin molecule, and the capacity of ACT constructs to penetrate into cell cytosol and deliver the epitope into the major histocompatibility complex (MHC) class I antigen processing and presentation pathway was examined. While all six constructs bearing the epitope within the Hly portion of ACT failed to deliver the epitope to the MHC class I molecules, all four toxoids with inserts within different permissive sites in the AC domain efficiently delivered the epitope into this cytosolic pathway, giving rise to stimulation of a specific CD8+ T-cell hybridoma. The results suggest that, in contrast to the AC domain, the hemolysin moiety of ACT does not reach the cytosolic entry of the MHC class I pathway.

Delivery of Multiple Epitopes by Recombinant Detoxified Adenylate Cyclase of Bordetella pertussis Induces Protective Antiviral Immunity

ABSTRACT CyaA, the adenylate cyclase toxin from Bordetella pertussis, can deliver its N-terminal catalytic domain into the cytosol of a large number of eukaryotic cells and particularly into professional antigen-presenting cells. We have previously identified within the primary structure of CyaA several permissive sites at which insertion of peptides does not alter the ability of the toxin to enter cells. This property has been exploited to design recombinant CyaA toxoids capable of delivering major histocompatibility complex (MHC) class I-restricted CD8+ T-cell epitopes into antigen-presenting cells and to induce specific CD8+cytotoxic T-lymphocyte (CTL) responses in vivo. Here we have explored the capacity of the CyaA vector carrying several different CD8+ T-cell epitopes to prime multiple CTL responses. The model vaccine consisted of a polyepitope made of three CTL epitopes from lymphocytic choriomeningitis virus (LCMV), the V3 region of human immunodeficiency virus gp120, and chicken ovalbumin, inserted at three different sites of the catalytic domain of genetically detoxified CyaA. Each of these epitopes was processed on delivery by CyaA and presented in vitro to specific T-cell hybridomas. Immunization of mice by CyaA toxoids carrying the polyepitope lead to the induction of specific CTL responses for each of the three epitopes, as well as to protection against a lethal viral challenge. Moreover, mice primed against the vector by mock CyaA or a recombinant toxoid were still able to develop strong CTL responses after subsequent immunization with a recombinant CyaA carrying a foreign CD8+ CTL epitope. These results highlight the potency of the adenylate cyclase vector for induction of protective CTL responses with multiple specificity and/or broad MHC restriction.

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.

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.

Oligomerization is involved in pore formation by Bordetella adenylate cyclase toxin

The Bordetella adenylate cyclase‐hemolysin (CyaA, ACT, or AC‐Hly) is a multifunctional toxin. Simultaneously with promoting calcium ion entry, CyaA delivers into host cells an adenylate cyclase enzyme (AC) and permeabilizes cell membrane by forming small cation‐selective pores. Indirect evidence suggested that these two activities were accomplished by different membraneinserted CyaA conformers, one acting as an AC‐delivering monomer and the other as an uncharacterized poreforming oligomer. We tested this model by directly detecting toxin oligomers in cell membrane and by assessing oligomerization of specific mutants with altered poreforming properties. CyaA oligomers were revealed in sheep erythrocyte membranes by immunogold labeling and directly demonstrated by pulldown of membraneinserted CyaA together with biotinylated CyaAAC‐ toxoid. Membrane oligomers of CyaAcould also be resolved by nondenaturing electrophoresis of mild detergent extracts of erythrocytes. Furthermore, CyaA mutants exhibiting enhanced (E581K) or reduced (E570K+E581P) specific hemolytic and pore‐forming activity were found to exhibit also a correspondingly enhanced or reduced propensity to form oligomers in erythrocyte membranes. On the other hand, processed CyaA, with the AC domain cleaved off by erythrocyte proteases, was detected only in a monomeric form excluded from the oligomers of unprocessed CyaA. These results provide the first direct evidence that oligomerization is involved in formation of CyaApores in target membranes and that translocation of the AC domain across cell membrane may be accomplished by monomelic CyaA.—Vojtova‐Vodolanova, J., Basler, M., Osicka, R., Knapp, O., Maier, E., Cerny, J., Benada, O., Benz, R., Sebo, P. Oligomerization is involved in pore formation by Bordetella adenylate cyclase toxin. FASEB J. 23, 2831–2843 (2009). www.fasebj.org

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.