A conserved tryptophan in the acylated segment of RTX toxins controls their ß2 integrin-independent cell penetration.

The acylated Repeats in ToXins (RTX) leukotoxins, the adenylate cyclase toxin (CyaA) or α-hemolysin (HlyA), bind ß2 integrins of leukocytes but also penetrate cells lacking these receptors. We show that the indoles of conserved tryptophans in the acylated segments, W876 of CyaA and W579 of HlyA, are crucial for ß2 integrin-independent membrane penetration. Substitutions of W876 by aliphatic or aromatic residues did not affect acylation, folding, or the activities of CyaA W876L/F/Y variants on cells expressing high amounts of the ß2 integrin CR3. However, toxin activity of CyaA W876L/F/Y on cells lacking CR3 was strongly impaired. Similarly, a W579L substitution selectively reduced HlyA W579L cytotoxicity towards cells lacking ß2 integrins. Intriguingly, the W876L/F/Y substitutions increased the thermal stability (Tm) of CyaA by 4 to 8 °C but locally enhanced the accessibility to deuteration of the hydrophobic segment and of the interface of the two acylated loops. W876Q substitution (showing no increase in Tm), or combination of W876F with a cavity-filling V822M substitution (this combination decreasing the Tm closer to that of CyaA), yielded a milder defect of toxin activity on erythrocytes lacking CR3. Furthermore, the activity of CyaA on erythrocytes was also selectively impaired when the interaction of the pyrrolidine of P848 with the indole of W876 was ablated. Hence, the bulky indoles of residues W876 of CyaA, or W579 of HlyA, rule the local positioning of the acylated loops and enable a membrane-penetrating conformation in the absence of RTX toxin docking onto the cell membrane by ß2 integrins.

Bordetella parapertussis adenylate cyclase toxin promotes the bacterial survival to the encounter with macrophages.

B. parapertussis is a whooping cough etiological agent, whose incidence in the population has increased remarkably. Virulence factors involved in the bacterial infection, however, remain poorly investigated. We here studied the role of adenylate cyclase (CyaA), the main toxin of B. parapertussis, in the outcome of the bacterial interaction with macrophages. Our results showed that B. parapertussis CyaA intoxicates human macrophages, prevents bacterial phagocytosis and precludes phago-lysosomal fusion eventually promoting the bacterial survival to the encounter with these immune cells. Accordingly, we found that B. parapertussis CyaA induces the transcriptional downregulation of host genes encoding for antimicrobial peptides, proteins involved in bacterial intracellular killing, and the pro-inflammatory cytokine TNF-α, while induces the upregulation of the anti-inflammatory cytokine IL-10. Together with previous reports suggesting a protective role of B. parapertussis CyaA against neutrophils bactericidal activity, the results of this study suggest a central role of CyaA in B. parapertussis immune evasion and persistence.

Phospholipase A activity of adenylate cyclase toxin mediates translocation of its adenylate cyclase domain.

Adenylate cyclase toxin (ACT or CyaA) plays a crucial role in respiratory tract colonization and virulence of the whooping cough causative bacterium Bordetella pertussis Secreted as soluble protein, it targets myeloid cells expressing the CD11b/CD18 integrin and on delivery of its N-terminal adenylate cyclase catalytic domain (AC domain) into the cytosol, generates uncontrolled toxic levels of cAMP that ablates bactericidal capacities of phagocytes. Our study deciphers the fundamentals of the heretofore poorly understood molecular mechanism by which the ACT enzyme domain directly crosses the host cell membrane. By combining molecular biology, biochemistry, and biophysics techniques, we discover that ACT has intrinsic phospholipase A (PLA) activity, and that such activity determines AC translocation. Moreover, we show that elimination of the ACT-PLA activity abrogates ACT toxicity in macrophages, particularly at toxin concentrations close to biological reality of bacterial infection. Our data support a molecular mechanism in which in situ generation of nonlamellar lysophospholipids by ACT-PLA activity into the cell membrane would form, likely in combination with membrane-interacting ACT segments, a proteolipidic toroidal pore through which AC domain transfer could directly take place. Regulation of ACT-PLA activity thus emerges as novel target for therapeutic control of the disease.

Bordetella pertussis Adenylate Cyclase Toxin Disrupts Functional Integrity of Bronchial Epithelial Layers.

The airway epithelium restricts the penetration of inhaled pathogens into the underlying tissue and plays a crucial role in the innate immune defense against respiratory infections. The whooping cough agent, Bordetella pertussis, adheres to ciliated cells of the human airway epithelium and subverts its defense functions through the action of secreted toxins and other virulence factors. We examined the impact of B. pertussis infection and of adenylate cyclase toxin-hemolysin (CyaA) action on the functional integrity of human bronchial epithelial cells cultured at the air-liquid interface (ALI). B. pertussis adhesion to the apical surface of polarized pseudostratified VA10 cell layers provoked a disruption of tight junctions and caused a drop in transepithelial electrical resistance (TEER). The reduction of TEER depended on the capacity of the secreted CyaA toxin to elicit cAMP signaling in epithelial cells through its adenylyl cyclase enzyme activity. Both purified CyaA and cAMP-signaling drugs triggered a decrease in the TEER of VA10 cell layers. Toxin-produced cAMP signaling caused actin cytoskeleton rearrangement and induced mucin 5AC production and interleukin-6 (IL-6) secretion, while it inhibited the IL-17A-induced secretion of the IL-8 chemokine and of the antimicrobial peptide beta-defensin 2. These results indicate that CyaA toxin activity compromises the barrier and innate immune functions of Bordetella-infected airway epithelia.

Bordetella adenylate cyclase toxin interacts with filamentous haemagglutinin to inhibit biofilm formation in vitro.

Bordetella pertussis, the causative agent of whooping cough, secretes and releases adenylate cyclase toxin (ACT), which is a protein bacterial toxin that targets host cells and disarms immune defenses. ACT binds filamentous haemagglutinin (FHA), a surface-displayed adhesin, and until now, the consequences of this interaction were unknown. A B. bronchiseptica mutant lacking ACT produced more biofilm than the parental strain; leading Irie et al. to propose the ACT-FHA interaction could be responsible for biofilm inhibition. Here we characterize the physical interaction of ACT with FHA and provide evidence linking that interaction to inhibition of biofilm in vitro. Exogenous ACT inhibits biofilm formation in a concentration-dependent manner and the N-terminal catalytic domain of ACT (AC domain) is necessary and sufficient for this inhibitory effect. AC Domain interacts with the C-terminal segment of FHA with ∼650 nM affinity. ACT does not inhibit biofilm formation by Bordetella lacking the mature C-terminal domain (MCD), suggesting the direct interaction between AC domain and the MCD is required for the inhibitory effect. Additionally, AC domain disrupts preformed biofilm on abiotic surfaces. The demonstrated inhibition of biofilm formation by a host-directed protein bacterial toxin represents a novel regulatory mechanism and identifies an unprecedented role for ACT.

Modulation of Pertussis and Adenylate Cyclase Toxins by Sigma Factor RpoE in Bordetella pertussis.

Bordetella pertussis is a human pathogen that can infect the respiratory tract and cause the disease known as whooping cough. B. pertussis uses pertussis toxin (PT) and adenylate cyclase toxin (ACT) to kill and modulate host cells to allow the pathogen to survive and persist. B. pertussis encodes many uncharacterized transcription factors, and very little is known about their functions. RpoE is a sigma factor which, in other bacteria, responds to oxidative, heat, and other environmental stresses. RseA is a negative regulator of RpoE that sequesters the sigma factor to regulate gene expression based on conditions. In B. pertussis, deletion of the rseA gene results in high transcriptional activity of RpoE and large amounts of secretion of ACT. By comparing parental B. pertussis to an rseA gene deletion mutant (PM18), we sought to characterize the roles of RpoE in virulence and determine the regulon of genes controlled by RpoE. Despite high expression of ACT, the rseA mutant strain did not infect the murine airway as efficiently as the parental strain and PM18 was killed more readily when inside phagocytes. RNA sequencing analysis was performed and 263 genes were differentially regulated by RpoE, and surprisingly, the rseA mutant strain where RpoE activity was elevated expressed very little pertussis toxin. Western blots and proteomic analysis corroborated the inverse relationship of PT to ACT expression in the high-RpoE-activity rseA deletion strain. Our data suggest that RpoE can modulate PT and ACT expression indirectly through unidentified mechanisms in response to conditions.

Acylation of the Bordetella pertussis CyaA-hemolysin: Functional implications for efficient membrane insertion and pore formation.

Previously, the ~130-kDa CyaA-hemolysin domain (CyaA-Hly) from Bordetella pertussis co-expressed with CyaC-acyltransferase in Escherichia coli was demonstrated to be palmitoylated at Lys983 and thus activated its hemolytic activity against target erythrocytes. Here, we report the functional importance of Lys983-palmitoylation for membrane insertion and pore formation of CyaA-Hly. Intrinsic fluorescence emissions of both non-acylated CyaA-Hly (NA/CyaA-Hly) and CyaA-Hly were indistinguishable, suggesting no severe conformational change upon acylation at Lys983. Following pre-incubation of sheep erythrocytes with NA/CyaA-Hly, there was a drastic decrease in CyaA-Hly-induced hemolysis. Direct interactions between NA/CyaA-Hly and target erythrocyte membranes were validated via membrane-binding assays along with Western blotting, suggestive of acylation-independent capability of NA/CyaA-Hly to interact with erythrocyte membranes. As compared with CyaA-Hly, NA/CyaA-Hly displayed a slower rate of incorporation into DOPC:DOPE:Ch or DiPhyPC bilayers under symmetrical conditions (1M KCl, 10mM HEPES, pH7.4) and formed channels exhibiting different conductance. Further analysis revealed that channel-open lifetime in DOPC:DOPE:Ch bilayers of NA/CyaA-Hly was much shorter than that of the acylated form, albeit slightly shorter lifetime found in DiPhyPC bilayers. Sequence alignments of the Lys983-containing CyaA-segment with those of related RTX-cytolysins revealed a number of highly conserved hydrophobic residues and a Lys/Arg cluster that is predicted be important for toxin-membrane interactions. Altogether, our data disclosed that the Lys983-linked palmitoyl group is not directly involved in either binding to target erythrocyte membranes or toxin-induced channel conductivity, but rather required for efficient membrane insertion and pore formation of the acylated CyaA-Hly domain.

Zn2+-dependent autocatalytic activity of the Bordetella pertussis CyaA-hemolysin.

Proteolytic degradation of the ∼100-kDa isolated RTX (Repeat-in-ToXin) subdomain (CyaA-RTX) of the Bordetella pertussis CyaA-hemolysin (CyaA-Hly) was evidently detected upon solely-prolonged incubation. Here, a truncated CyaA-Hly fragment (CyaA-HP/BI) containing hydrophobic and acylation regions connected with the first RTX block (BI1015-1088) was constructed as a putative precursor for investigating its potential autocatalysis. The 70-kDa His-tagged CyaA-HP/BI fragment which was over-expressed in Escherichia coli as insoluble aggregate was entirely solubilized with 4 M urea. After re-naturation in a Ni2+-NTA affinity column, the purified-refolded CyaA-HP/BI fragment in HEPES buffer (pH 7.4) supplemented with 2 mM CaCl2 was completely degraded upon incubation at 37 °C for 3 h. Addition of 1,10-phenanthroline‒an inhibitor of Zn2+-dependent metalloproteases markedly reduced the extent of degradation for CyaA-HP/BI and CyaA-RTX, but the degradative effect was clearly enhanced by addition of 100 mM ZnCl2. Structural analysis of a plausible CyaA-HP/BI model revealed a potential Zn2+-binding His-Asp cluster located between the acylation region and RTX-BI1015-1088. Moreover, Arg997‒one of the identified cleavage sites of the CyaA-RTX fragment was located in close proximity to the Zn2+-binding catalytic site. Overall results demonstrated for the first time that the observed proteolysis of CyaA-HP/BI and CyaA-RTX fragments is conceivably due to their Zn2+-dependent autocatalytic activity.

A fortunate journey on uneven grounds.

I was surprised to be invited to write a prefatory chapter for the Annual Review of Microbiology. Indeed, I did not feel that I belonged to that class of eminent scientists who had written such chapters. Perhaps it is because I am a kind of mutant: In spite of having experienced war, both German and Soviet occupations, repeated bombardments, dictatorships, and a revolution, I managed nonetheless to engage in scientific research, thus realizing a childhood dream. After having obtained my Doctor Rerum Naturalium degree in Budapest, Hungary, I was fortunate to meet Jacques Monod at the Pasteur Institute, and this became a turning point in my scientific career. In his laboratory, I contributed to the definition of the lactose operon promoter, uncovered intracistronic complementation in ß-galactosidase, and investigated the role of cAMP in Escherichia coli. In my own laboratory, together with many gifted students and collaborators, I studied the role of adenylate cyclase in bacterial virulence. This allowed the engineering of recombinant adenylate cyclase toxin from Bordetella pertussis for the development of protective and therapeutic vaccines.

Pore-formation by adenylate cyclase toxoid activates dendritic cells to prime CD8+ and CD4+ T cells.

The adenylate cyclase toxin-hemolysin (CyaA) of Bordetella pertussis is a bi-functional leukotoxin. It penetrates myeloid phagocytes expressing the complement receptor 3 and delivers into their cytosol its N-terminal adenylate cyclase enzyme domain (~400 residues). In parallel, ~1300 residue-long RTX hemolysin moiety of CyaA forms cation-selective pores and permeabilizes target cell membrane for efflux of cytosolic potassium ions. The non-enzymatic CyaA-AC(-) toxoid, has repeatedly been successfully exploited as an antigen delivery tool for stimulation of adaptive T-cell immune responses. We show that the pore-forming activity confers on the CyaA-AC(-) toxoid a capacity to trigger Toll-like receptor and inflammasome signaling-independent maturation of CD11b-expressing dendritic cells (DC). The DC maturation-inducing potency of mutant toxoid variants in vitro reflected their specifically enhanced or reduced pore-forming activity and K(+) efflux. The toxoid-induced in vitro phenotypic maturation of DC involved the activity of mitogen activated protein kinases p38 and JNK and comprised increased expression of maturation markers, interleukin 6, chemokines KC and LIX and granulocyte-colony-stimulating factor secretion, prostaglandin E2 production and enhancement of chemotactic migration of DC. Moreover, i.v. injected toxoids induced maturation of splenic DC in function of their cell-permeabilizing capacity. Similarly, the capacity of DC to stimulate CD8(+) and CD4(+) T-cell responses in vitro and in vivo was dependent on the pore-forming activity of CyaA-AC(-). This reveals a novel self-adjuvanting capacity of the CyaA-AC(-) toxoid that is currently under clinical evaluation as a tool for delivery of immunotherapeutic anti-cancer CD8(+) T-cell vaccines into DC.

Allosteric activation of Bordetella pertussis adenylyl cyclase by calmodulin: molecular dynamics and mutagenesis studies.

Adenylyl cyclase (AC) toxin is an essential toxin that allows Bordetella pertussis to invade eukaryotic cells, where it is activated after binding to calmodulin (CaM). Based on the crystal structure of the AC catalytic domain in complex with the C-terminal half of CaM (C-CaM), our previous molecular dynamics simulations (Selwa, E., Laine, E., and Malliavin, T. (2012) Differential role of calmodulin and calcium ions in the stabilization of the catalytic domain of adenyl cyclase CyaA from Bordetella pertussis. Proteins 80, 1028­1040) suggested that three residues (i.e. Arg(338), Asn(347), and Asp(360)) might be important for stabilizing the AC/CaM interaction. These residues belong to a loop-helix-loop motif at the C-terminal end of AC, which is located at the interface between CaM and the AC catalytic loop. In the present study, we conducted the in silico and in vitro characterization of three AC variants, where one (Asn(347); ACm1A), two (Arg(338) and Asp(360); ACm2A), or three residues (Arg(338), Asn(347), and Asp(360); ACm3A) were substituted with Ala. Biochemical studies showed that the affinities of ACm1A and ACm2A for CaM were not affected significantly, whereas that of ACm3A was reduced dramatically. To understand the effects of these modifications, molecular dynamics simulations were performed based on the modified proteins. The molecular dynamics trajectories recorded for the ACm3AC-CaM complex showed that the calcium-binding loops of C-CaM exhibited large fluctuations, which could be related to the weakened interaction between ACm3A and its activator. Overall, our results suggest that the loop-helix-loop motif at the C-terminal end of AC is crucial during CaM binding for stabilizing the AC catalytic loop in an active configuration.

Heterogeneity of Bordetella bronchiseptica adenylate cyclase (cyaA) RTX domain.

Bordetella bronchiseptica is a widespread pathogen, with a broad host range, occasionally including humans. Diverse virulence factors (adhesins, toxins) allow its adaptation to its host, but this property of the adenylate cyclase (cyaA) toxin is not well understood. In this study, we analyzed the repeats-in-toxin domain of B. bronchiseptica cyaA with PCR, followed by restriction fragment length analysis. Of ninety-two B. bronchiseptica strains collected from different hosts and geographic regions, 72 (78.3 %) carried cyaA and four RFLP types (A-D) were established using NarI and SalI. However, in 20 strains, cyaA was replaced with a peptide transport protein operon. A phylogenetic tree based on partial nucleotide sequences of cyaA revealed that group 2 contains strains of specifically human origin, whereas subgroup 1a contains all but one of the strains from pigs. The human strains showed many PCR-RFLP and sequence variants, confirming the clonal population structure of B. bronchiseptica.

Mutation in the ß-hairpin of the Bordetella pertussis adenylate cyclase toxin modulates N-lobe conformation in calmodulin.

Bordetella pertussis, causative agent of whooping cough, produces an adenylate cyclase toxin (CyaA) that is an important virulence factor. In the host cell, the adenylate cyclase domain of CyaA (CyaA-ACD) is activated upon association with calmodulin (CaM), an EF-hand protein comprised of N- and C-lobes (N-CaM and C-CaM, respectively) connected by a flexible tether. Maximal CyaA-ACD activation is achieved through its binding to both lobes of intact CaM, but the structural mechanisms remain unclear. No high-resolution structure of the intact CaM/CyaA-ACD complex is available, but crystal structures of isolated C-CaM bound to CyaA-ACD shed light on the molecular mechanism by which this lobe activates the toxin. Previous studies using molecular modeling, biochemical, and biophysical experiments demonstrate that CyaA-ACD's ß-hairpin participates in site-specific interactions with N-CaM. In this study, we utilize nuclear magnetic resonance (NMR) spectroscopy to probe the molecular association between intact CaM and CyaA-ACD. Our results indicate binding of CyaA-ACD to CaM induces large conformational perturbations mapping to C-CaM, while substantially smaller structural changes are localized primarily to helices I, II, and IV, and the metal-binding sites in N-CaM. Site-specific mutations in CyaA-ACD's ß-hairpin structurally modulate N-CaM, resulting in conformational perturbations in metal binding sites I and II, while no significant structural modifications are observed in C-CaM. Moreover, dynamic light scattering (DLS) analysis reveals that mutation of the ß-hairpin results in a decreased hydrodynamic radius (Rh) and reduced thermal stability in the mutant complex. Taken together, our data provide new structural insights into the ß-hairpin's role in stabilizing interactions between CyaA-ACD and N-CaM.

Bordetella filamentous hemagglutinin and adenylate cyclase toxin interactions on the bacterial surface are consistent with FhaB-mediated delivery of ACT to phagocytic cells.

Bordetella species that cause respiratory infections in mammals include B. pertussis, which causes human whooping cough, and B. bronchiseptica, which infects nearly all mammals. Both bacterial species produce filamentous hemagglutinin (FhaB) and adenylate cyclase toxin (ACT), prominent surface-associated and secreted virulence factors that contribute to persistence in the lower respiratory tract by inhibiting clearance by phagocytic cells. FhaB and ACT proteins interact with themselves, each other, and host cells. Using immunoblot analyses, we showed that ACT binds to FhaB on the bacterial surface before it can be detected in culture supernatants. We determined that SphB1, a surface protease identified based on its requirement for FhaB cleavage, is also required for ACT cleavage, and we determined that the presence of ACT blocks SphB1-dependent and -independent cleavage of FhaB, but the presence of FhaB does not affect SphB1-dependent cleavage of ACT. The primary SphB1-dependent cleavage site on ACT is proximal to ACT's active site, in a region that is critical for ACT activity. We also determined that FhaB-bound ACT on the bacterial surface can intoxicate host cells producing CR3, the receptor for ACT. In addition to increasing our understanding of FhaB, ACT, and FhaB-ACT interactions on the Bordetella surface, our data are consistent with a model in which FhaB functions as a novel toxin delivery system by binding to ACT and allowing its release upon binding of ACT to its receptor, CR3, on phagocytic cells.IMPORTANCEBacteria need to control the variety, abundance, and conformation of proteins on their surface to survive. Members of the Gram-negative bacterial genus Bordetella include B. pertussis, which causes whooping cough in humans, and B. bronchiseptica, which causes respiratory infections in a broad range of mammals. These species produce two prominent virulence factors, the two-partner secretion (TPS) effector FhaB and adenylate cyclase toxin (ACT), that interact with themselves, each other, and host cells. Here, we determined that ACT binds FhaB on the bacterial surface before being detected in culture supernatants and that ACT bound to FhaB can be delivered to eukaryotic cells. Our data are consistent with a model in which FhaB delivers ACT specifically to phagocytic cells. This is the first report of a TPS system facilitating the delivery of a separate polypeptide toxin to target cells and expands our understanding of how TPS systems contribute to bacterial pathogenesis.

Modification of the RTX domain cap by acyl chains of adapted length rules the formation of functional hemolysin pores.

The acylated pore-forming Repeats in ToXin (RTX) cytolysins α-hemolysin (HlyA) and adenylate cyclase toxin (CyaA) preferentially bind to ß2 integrins of myeloid leukocytes but can also promiscuously bind and permeabilize cells lacking the ß2 integrins. We constructed a HlyA1-563/CyaA860-1706 chimera that was acylated either by the toxin-activating acyltransferase CyaC, using sixteen carbon-long (C16) acyls, or by the HlyC acyltransferase using fourteen carbon-long (C14) acyls. Cytolysin assays with the C16- or C14-acylated HlyA/CyaA chimeric toxin revealed that the RTX domain of CyaA can functionally replace the RTX domain of HlyA only if it is modified by C16-acyls on the Lys983 residue of CyaA. The C16-monoacylated HlyA/CyaA chimera was as pore-forming and cytolytic as native HlyA, whereas the C14-acylated chimera exhibited very low pore-forming activity. Hence, the capacity of the RTX domain of CyaA to support the insertion of the N-terminal pore-forming domain into the target cell membrane, and promote formation of toxin pores, strictly depends on the modification of the Lys983 residue by an acyl chain of adapted length.

Identification of a region that assists membrane insertion and translocation of the catalytic domain of Bordetella pertussis CyaA toxin.

The adenylate cyclase (CyaA) toxin, one of the virulence factors secreted by Bordetella pertussis, the pathogenic bacteria responsible for whooping cough, plays a critical role in the early stages of respiratory tract colonization by this bacterium. The CyaA toxin is able to invade eukaryotic cells by translocating its N-terminal catalytic domain directly across the plasma membrane of the target cells, where, activated by endogenous calmodulin, it produces supraphysiological levels of cAMP. How the catalytic domain is transferred from the hydrophilic extracellular medium into the hydrophobic environment of the membrane and then to the cell cytoplasm remains an unsolved question. In this report, we have characterized the membrane-interacting properties of the CyaA catalytic domain. We showed that a protein covering the catalytic domain (AC384, encompassing residues 1-384 of CyaA) displayed no membrane association propensity. However, a longer polypeptide (AC489), encompassing residues 1-489 of CyaA, exhibited the intrinsic property to bind to membranes and to induce lipid bilayer destabilization. We further showed that deletion of residues 375-485 within CyaA totally abrogated the toxin's ability to increase intracellular cAMP in target cells. These results indicate that, whereas the calmodulin dependent enzymatic domain is restricted to the amino-terminal residues 1-384 of CyaA, the membrane-interacting, translocation-competent domain extends up to residue 489. This thus suggests an important role of the region adjacent to the catalytic domain of CyaA in promoting its interaction with and its translocation across the plasma membrane of target cells.

An improved recombination-based in vivo expression technology-like reporter system reveals differential cyaA gene activation in Bordetella species.

Bordetella pertussis and Bordetella bronchiseptica rely on the global two-component regulatory system BvgAS to control expression of distinct phenotypic phases. In the Bvg(-) phase, expression of vrg genes, including those required for motility in B. bronchiseptica, is activated and genes encoding virulence factors are not expressed. Conversely, in the Bvg(+) phase, genes encoding virulence factors are highly expressed while genes necessary for motility are repressed. Although several genetic analyses have demonstrated the importance of the Bvg(+) phase during respiratory infection, Bvg-regulated gene activation in B. bronchiseptica has not been investigated in vivo. To address this, we developed a plasmid, pGFLIP, that encodes a sensitive Flp recombinase-based fluorescent reporter system able to document gene activation both in vitro and in vivo. Using pGFLIP, we demonstrated that cyaA, considered to be a "late" Bvg(+) phase gene, is activated substantially earlier in B. bronchiseptica than B. pertussis following a switch from Bvg(-) to Bvg(+) phase conditions. We show that the altered activation of cyaA is not due to differences in the cyaA promoter or in the bvgAS alleles of B. bronchiseptica compared to B. pertussis, but appears to be species specific. Finally, we used pGFLIP to show that flaA remains repressed during infection, confirming that B. bronchiseptica does not modulate to the Bvg(-) phase in vivo.

The bacterial two-hybrid system based on adenylate cyclase reconstitution in Escherichia coli.

The bacterial two-hybrid system based on the reconstitution of adenylate cyclase in Escherichia coli (BACTH) was described 14years ago (Karimova, Pidoux, Ullmann, and Ladant, 1998, PNAS, 95:5752). For microbiologists, it is a practical and powerful alternative to the use of the widely spread yeast two-hybrid technology for testing protein-protein interactions. In this review, we aim at giving the reader clear and most importantly simple instructions that should break any reticence to try the technique. Yet, we also add recommendations in the use of the system, related to its specificities. Finally, we expose the advantages and disadvantages of the technique, and review its diverse applications in the literature, which should help in deciding if it is the appropriate method to choose for the case at hand.

Delivery of large heterologous polypeptides across the cytoplasmic membrane of antigen-presenting cells by the Bordetella RTX hemolysin moiety lacking the adenylyl cyclase domain.

The Bordetella adenylate cyclase toxin-hemolysin (CyaA; also called ACT or AC-Hly) targets CD11b-expressing phagocytes and translocates into their cytosol an adenylyl cyclase (AC) that hijacks cellular signaling by conversion of ATP to cyclic AMP (cAMP). Intriguingly, insertion of large passenger peptides removes the enzymatic activity but not the cell-invasive capacity of the AC domain. This has repeatedly been exploited for delivery of heterologous antigens into the cytosolic pathway of CD11b-expressing dendritic cells by CyaA/AC(-) toxoids, thus enabling their processing and presentation on major histocompatibility complex (MHC) class I molecules to cytotoxic CD8(+) T lymphocytes (CTLs). We produced a set of toxoids with overlapping deletions within the first 371 residues of CyaA and showed that the structure of the AC enzyme does not contain any sequences indispensable for its translocation across target cell membrane. Moreover, replacement of the AC domain (residues 1 to 371) with heterologous polypeptides of 40, 146, or 203 residues yielded CyaAΔAC constructs that delivered passenger CTL epitopes into antigen-presenting cells (APCs) and induced strong antigen-specific CD8(+) CTL responses in vivo in mice and ex vivo in human peripheral blood mononuclear cell cultures. This shows that the RTX (repeats in toxin) hemolysin moiety, consisting of residues 374 to 1706 of CyaA, harbors all structural information involved in translocation of the N-terminal AC domain across target cell membranes. These results decipher the extraordinary capacity of the AC domain of CyaA to transport large heterologous cargo polypeptides into the cytosol of CD11b(+) target cells and pave the way for the construction of CyaAΔAC-based polyvalent immunotherapeutic T cell vaccines.

Contribution of Bordetella filamentous hemagglutinin and adenylate cyclase toxin to suppression and evasion of interleukin-17-mediated inflammation.

Bordetella pertussis and Bordetella bronchiseptica establish respiratory infections with notorious efficiency. Our previous studies showed that the fhaB genes of B. pertussis and B. bronchiseptica, which encode filamentous hemagglutinin (FHA), are functionally interchangeable and provided evidence that FHA-deficient B. bronchiseptica induces more inflammation in the lungs of mice than wild-type B. bronchiseptica. We show here that the robust inflammatory response to FHA-deficient B. bronchiseptica is characterized by the early and sustained influx of interleukin-17 (IL-17)-positive neutrophils and macrophages and, at 72 h postinoculation, IL-17-positive CD4(+) T cells, suggesting that FHA allows the bacteria to suppress the development of an IL-17-mediated inflammatory response. We also show that the cyaA genes of B. pertussis and B. bronchiseptica, which encode adenylate cyclase toxin (ACT), are functionally interchangeable and that ACT, specifically its catalytic activity, is required for B. bronchiseptica to resist phagocytic clearance but is neither required for nor inhibitory of the induction of inflammation if bacteria are present in numbers sufficient to persist during the first 3 days postinoculation. Incubation of bone marrow-derived macrophages with a ΔcyaA strain caused decreased production of IL-1ß and increased production of tumor necrosis factor alpha (TNF-α) and IL-12, while incubation with a ΔcyaA ΔfhaB strain caused increased production of IL-23. These data suggest that FHA and ACT both contribute to suppress the recruitment of neutrophils and the development of an IL-17-mediated immune response. To our knowledge, this is the first demonstration of a microbial pathogen suppressing IL-17-mediated inflammation in vivo as a strategy to evade innate immunity.