Halogen-Dance-Based Synthesis of Phosphonomethoxyethyl (PME) Substituted 2-Aminothiazoles as Potent Inhibitors of Bacterial Adenylate Cyclases.

A series of acyclic nucleoside phosphonates (ANPs) was designed as inhibitors of bacterial adenylate cyclases (ACs), where adenine was replaced with 2-amino-4-arylthiazoles. The target compounds were prepared using the halogen dance reaction. Final AC inhibitors were evaluated in cell-based assays (prodrugs) and cell-free assays (phosphono diphosphates). Novel ANPs were potent inhibitors of adenylate cyclase toxin (ACT) from Bordetella pertussis and edema factor (EF) from Bacillus anthracis, with substantial selectivity over mammalian enzymes AC1, AC2, and AC5. Six of the new ANPs were more potent or equipotent ACT inhibitors (IC50 =9-18 nM), and one of them was more potent EF inhibitor (IC50 =12 nM), compared to adefovir diphosphate (PMEApp) with IC50 =18 nM for ACT and IC50 =36 nM for EF. Thus, these compounds represent the most potent ACT/EF inhibitors based on ANPs reported to date. The potency of the phosphonodiamidates to inhibit ACT activity in J774A.1 macrophage cells was somewhat weaker, where the most potent derivative had IC50 =490 nM compared to IC50 =150 nM of the analogous adefovir phosphonodiamidate. The results suggest that more efficient type of phosphonate prodrugs would be desirable to increase concentrations of the ANP-based active species in the cells in order to proceed with the development of ANPs as potential antitoxin therapeutics.

Acyclic nucleoside phosphonates with 2-aminothiazole base as inhibitors of bacterial and mammalian adenylate cyclases.

A series of novel acyclic nucleoside phosphonates (ANPs) was synthesized as potential adenylate cyclase inhibitors, where the adenine nucleobase of adefovir (PMEA) was replaced with a 5-substituted 2-aminothiazole moiety. The design was based on the structure of MB05032, a potent and selective inhibitor of fructose 1,6-bisphosphatase and a good mimic of adenosine monophosphate (AMP). From the series of eighteen novel ANPs, which were prepared as phosphoroamidate prodrugs, fourteen compounds were potent (single digit micromolar or submicromolar) inhibitors of Bordetella pertussis adenylate cyclase toxin (ACT), mostly without observed cytotoxicity in J774A.1 macrophage cells. Selected phosphono diphosphates (nucleoside triphosphate analogues) were potent inhibitors of ACT (IC50 as low as 37 nM) and B. anthracis edema factor (IC50 as low as 235 nM) in enzymatic assays. Furthermore, several ANPs were found to be selective mammalian AC1 inhibitors in HEK293 cell-based assays (although with some associated cytotoxicity) and one compound exhibited selective inhibition of mammalian AC2 (only 12% of remaining adenylate cyclase activity) but no observed cytotoxicity. The mammalian AC1 inhibitors may represent potential leads in development of agents for treatment of human inflammatory and neuropathic pain.

Pharmacological targeting of host chaperones protects from pertussis toxin in vitro and in vivo.

Whooping cough is caused by Bordetella pertussis that releases pertussis toxin (PT) which comprises enzyme A-subunit PTS1 and binding/transport B-subunit. After receptor-mediated endocytosis, PT reaches the endoplasmic reticulum from where unfolded PTS1 is transported to the cytosol. PTS1 ADP-ribosylates G-protein α-subunits resulting in increased cAMP signaling. Here, a role of target cell chaperones Hsp90, Hsp70, cyclophilins and FK506-binding proteins for cytosolic PTS1-uptake is demonstrated. PTS1 specifically and directly interacts with chaperones in vitro and in cells. Specific pharmacological chaperone inhibition protects CHO-K1, human primary airway basal cells and a fully differentiated airway epithelium from PT-intoxication by reducing intracellular PTS1-amounts without affecting cell binding or enzyme activity. PT is internalized by human airway epithelium secretory but not ciliated cells and leads to increase of apical surface liquid. Cyclophilin-inhibitors reduced leukocytosis in infant mouse model of pertussis, indicating their promising potential for developing novel therapeutic strategies against whooping cough.

Bioengineering of Bordetella pertussis Adenylate Cyclase Toxin for Vaccine Development and Other Biotechnological Purposes.

The adenylate cyclase toxin, CyaA, is one of the key virulent factors produced by Bordetella pertussis, the causative agent of whooping cough. This toxin primarily targets innate immunity to facilitate bacterial colonization of the respiratory tract. CyaA exhibits several remarkable characteristics that have been exploited for various applications in vaccinology and other biotechnological purposes. CyaA has been engineered as a potent vaccine vehicle to deliver antigens into antigen-presenting cells, while the adenylate cyclase catalytic domain has been used to design a robust genetic assay for monitoring protein-protein interactions in bacteria. These two biotechnological applications are briefly summarized in this chapter.

Development of novel isatin-nicotinohydrazide hybrids with potent activity against susceptible/resistant Mycobacterium tuberculosis and bronchitis causing-bacteria.

Joining the global fight against Tuberculosis, the world's most deadly infectious disease, herein we present the design and synthesis of novel isatin-nicotinohydrazide hybrids (5a-m and 9a-c) as promising anti-tubercular and antibacterial agents. The anti-tubercular activity of the target hybrids was evaluated against drug-susceptible M. tuberculosis strain (ATCC 27294) where hybrids 5d, 5g and 5h were found to be as potent as INH with MIC = 0.24 µg/mL, also the activity was evaluated against Isoniazid/Streptomycin resistant M. tuberculosis (ATCC 35823) where compounds 5g and 5h showed excellent activity (MIC = 3.9 µg/mL). Moreover, the target hybrids were examined against six bronchitis causing-bacteria. Most derivatives exhibited excellent antibacterial activity. K. pneumonia emerged as the most sensitive strain with MIC range: 0.49-7.81 µg/mL. Furthermore, a molecular docking study has proposed DprE1 as a probable enzymatic target for herein reported isatin-nicotinohydrazide hybrids, and explored the binding interactions within the vicinity of DprE1 active site.

Susceptibility of primary human airway epithelial cells to Bordetella pertussis adenylate cyclase toxin in two- and three-dimensional culture conditions.

The human pathogen Bordetella pertussis targets the respiratory epithelium and causes whooping cough. Its virulence factor adenylate cyclase toxin (CyaA) plays an important role in the course of infection. Previous studies on the impact of CyaA on human epithelial cells have been carried out using cell lines derived from the airways or the intestinal tract. Here, we investigated the interaction of CyaA and its enzymatically inactive but fully pore-forming toxoid CyaA-AC- with primary human airway epithelial cells (hAEC) derived from different anatomical sites (nose and tracheo-bronchial region) in two-dimensional culture conditions. To assess possible differences between the response of primary hAEC and respiratory cell lines directly, we included HBEC3-KT in our studies. In comparative analyses, we studied the impact of both the toxin and the toxoid on cell viability, intracellular cAMP concentration and IL-6 secretion. We found that the selected hAEC, which lack CD11b, were differentially susceptible to both CyaA and CyaA-AC-. HBEC3-KT appeared not to be suitable for subsequent analyses. Since the nasal epithelium first gets in contact with airborne pathogens, we further studied the effect of CyaA and its toxoid on the innate immunity of three-dimensional tissue models of the human nasal mucosa. The present study reveals first insights in toxin-cell interaction using primary hAEC.

Preferential modification of CyaA-hemolysin by CyaC-acyltransferase through the catalytic Ser30-His33 dyad in esterolysis of palmitoyl-donor substrate devoid of acyl carrier proteins.

We previously demonstrated that the ~130-kDa CyaA-hemolysin domain (CyaA-Hly) from Bordetella pertussis co-expressed with CyaC-acyltransferase in Escherichia coli was acylated at Lys983 and thus activated its hemolytic activity. Here, attempts were made to provide greater insights into such toxin activation via fatty-acyl modification by CyaC-acyltransferase. Non-acylated CyaA-Hly (NA/CyaA-Hly) and CyaC were separately expressed in E. coli and subsequently purified by FPLC to near homogeneity. When effects of acyl-chain length were comparatively evaluated through CyaC-esterolysis using various p-nitrophenyl (pNP) derivatives, Michaelis-Menten steady-state kinetic parameters (KM and kcat) of CyaC-acyltransferase revealed a marked preference for myristoyl (C14:0) and palmitoyl (C16:0) substrates of which catalytic efficiencies (kcat/KM) were roughly the same (~1.5 × 103 s-1mM-1). However, pNP-palmitate (pNPP) gave the highest hemolytic activity of NA/CyaA-Hly after being acylated in vitro with a range of acyl-donor substrates. LC-MS/MS analysis confirmed such CyaC-mediated palmitoylation of CyaA-Hly occurring at Lys983, denoting no requirement of an acyl carrier protein (ACP). A homology-based CyaC structure inferred a role of a potential catalytic dyad of conserved Ser30 and His33 residues in substrate esterolysis. CyaC-ligand binding analysis via molecular docking corroborated high-affinity binding of palmitate with its carboxyl group oriented toward such a dyad. Ala-substitutions of each residue (S30A or H33A) caused a drastic decrease in kcat/KM of CyaC toward pNPP, and hence its catalytic malfunction through palmitoylation-dependent activation of NA/CyaA-Hly. Altogether, our present data evidently provide such preferential palmitoylation of CyaA-Hly by CyaC-acyltransferase through the enzyme Ser30-His33 nucleophile-activation dyad in esterolysis of palmitoyl-donor substrate, particularly devoid of a natural acyl-ACP donor.

Production of Highly Active Recombinant Dermonecrotic Toxin of Bordetella Pertussis.

Pathogenic Bordetella bacteria release a neurotropic dermonecrotic toxin (DNT) that is endocytosed into animal cells and permanently activates the Rho family GTPases by polyamination or deamidation of the glutamine residues in their switch II regions (e.g., Gln63 of RhoA). DNT was found to enable high level colonization of the nasal cavity of pigs by B. bronchiseptica and the capacity of DNT to inhibit differentiation of nasal turbinate bone osteoblasts causes atrophic rhinitis in infected pigs. However, it remains unknown whether DNT plays any role also in virulence of the human pathogen B. pertussis and in pathogenesis of the whooping cough disease. We report a procedure for purification of large amounts of LPS-free recombinant DNT that exhibits a high biological activity on cells expressing the DNT receptors Cav3.1 and Cav3.2. Electron microscopy and single particle image analysis of negatively stained preparations revealed that the DNT molecule adopts a V-shaped structure with well-resolved protein domains. These results open the way to structure-function studies on DNT and its interactions with airway epithelial layers.

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

Bordetella pertussis, the causative agent of whopping cough, produces an adenylate cyclase toxin (CyaA) that plays a key role in the host colonization by targeting innate immune cells which express CD11b/CD18, the cellular receptor of CyaA. CyaA is also able to invade non-phagocytic cells, via a unique entry pathway consisting in a direct translocation of its catalytic domain across the cytoplasmic membrane of the cells. Within the cells, CyaA is activated by calmodulin to produce high levels of cyclic adenosine monophosphate (cAMP) and alter cellular physiology. In this study, we explored the effects of CyaA toxin on the cellular and molecular structure remodeling of A549 alveolar epithelial cells. Using classical imaging techniques, biochemical and functional tests, as well as advanced cell mechanics method, we quantify the structural and functional consequences of the massive increase of intracellular cyclic AMP induced by the toxin: cell shape rounding associated to adhesion weakening process, actin structure remodeling for the cortical and dense components, increase in cytoskeleton stiffness, and inhibition of migration and repair. We also show that, at low concentrations (0.5 nM), CyaA could significantly impair the migration and wound healing capacities of the intoxicated alveolar epithelial cells. As such concentrations might be reached locally during B. pertussis infection, our results suggest that the CyaA, beyond its major role in disabling innate immune cells, might also contribute to the local alteration of the epithelial barrier of the respiratory tract, a hallmark of pertussis.

π-Helix controls activity of oxygen-sensing diguanylate cyclases.

The ability of organisms to sense and adapt to oxygen levels in their environment leads to changes in cellular phenotypes, including biofilm formation and virulence. Globin coupled sensors (GCSs) are a family of heme proteins that regulate diverse functions in response to O2 levels, including modulating synthesis of cyclic dimeric guanosine monophosphate (c-di-GMP), a bacterial second messenger that regulates biofilm formation. While GCS proteins have been demonstrated to regulate O2-dependent pathways, the mechanism by which the O2 binding event is transmitted from the globin domain to the cyclase domain is unknown. Using chemical cross-linking and subsequent liquid chromatography-tandem mass spectrometry, diguanylate cyclase (DGC)-containing GCS proteins from Bordetella pertussis (BpeGReg) and Pectobacterium carotovorum (PccGCS) have been demonstrated to form direct interactions between the globin domain and a middle domain π-helix. Additionally, mutation of the π-helix caused major changes in oligomerization and loss of DGC activity. Furthermore, results from assays with isolated globin and DGC domains found that DGC activity is affected by the cognate globin domain, indicating unique interactions between output domain and cognate globin sensor. Based on these studies a compact GCS structure, which depends on the middle domain π-helix for orienting the three domains, is needed for DGC activity and allows for direct sensor domain interactions with both middle and output domains to transmit the O2 binding signal. The insights from the present study improve our understanding of DGC regulation and provide insight into GCS signaling that may lead to the ability to rationally control O2-dependent GCS activity.

A LONELY GUY protein of Bordetella pertussis with unique features is related to oxidative stress.

The Gram-negative bacterium B. pertussis is the causative agent of whooping cough. This infection is re-emerging and new features related to Bordetella pathogenesis and microbiology could be relevant to defeat it. Therefore, we focused our attention on BP1253, a predicted exported protein from B. pertussis erroneously classified as lysine decarboxylase. We showed that BP1253 shares the highly conserved motif PGGxGTxxE and the key catalytic amino-acid residues with newly structurally characterized "LONELY GUY" (LOG) proteins. Biochemical studies have confirmed that this protein functions as a cytokinin-activating enzyme since it cleaves the N-glycosidic linkage between the base and the ribose, leading to the formation of free bases, which are the active form of plant hormones called cytokinins. Remarkably, BP1253 selectively binds monophosphate nucleotides such as AMP, GMP and CMP, showing a wider variety in binding capacity compared to other LOGs. Cytokinin production studies performed with B. pertussis have revealed 6-O-methylguanine to be the physiological product of BP1253 in agreement with the higher activity of the enzyme towards GMP. 6-O-methylguanine is likely to be responsible for the increased sensitivity of B. pertussis to oxidative stress. Although BP1253 has a primary sequence resembling the hexameric type-II LOGs, the dimeric state and the presence of specific amino-acids suggests that BP1253 can be classified as a novel type-II LOG. The discovery of a LOG along with its product 6-O-methylguanine in the human pathogen B. pertussis may lead to the discovery of unexplored functions of LOGs, broadening their role beyond plants.

Role of Major Toxin Virulence Factors in Pertussis Infection and Disease Pathogenesis.

Bordetella pertussis produces several toxins that affect host-pathogen interactions. Of these, the major toxins that contribute to pertussis infection and disease are pertussis toxin, adenylate cyclase toxin-hemolysin and tracheal cytotoxin. Pertussis toxin is a multi-subunit protein toxin that inhibits host G protein-coupled receptor signaling, causing a wide array of effects on the host. Adenylate cyclase toxin-hemolysin is a single polypeptide, containing an adenylate cyclase enzymatic domain coupled to a hemolysin domain, that primarily targets phagocytic cells to inhibit their antibacterial activities. Tracheal cytotoxin is a fragment of peptidoglycan released by B. pertussis that elicits damaging inflammatory responses in host cells. This chapter describes these three virulence factors of B. pertussis, summarizing background information and focusing on the role of each toxin in infection and disease pathogenesis, as well as their role in pertussis vaccination.

Regulated, sequential processing by multiple proteases is required for proper maturation and release of Bordetella filamentous hemagglutinin.

Filamentous hemagglutinin (FHA) is a critically important virulence factor produced by Bordetella species that cause respiratory infections in humans and other animals. It is also a prototypical member of the widespread two partner secretion (TPS) pathway family of proteins. First synthesized as a ~370 kDa protein called FhaB, its C-terminal ~1,200 amino acid 'prodomain' is removed during translocation to the cell surface via the outer membrane channel FhaC. Here, we identify CtpA as a periplasmic protease that is responsible for the regulated degradation of the prodomain and for creation of an intermediate polypeptide that is cleaved by the autotransporter protease SphB1 to generate FHA. We show that the central prodomain region is required to initiate degradation of the prodomain and that CtpA degrades the prodomain after a third, unidentified protease (P3) first removes the extreme C-terminus of the prodomain. Stepwise proteolysis by P3, CtpA and SphB1 is required for maturation of FhaB, release of FHA into the extracellular milieu, and full function in vivo. These data support a substantially updated model for the mechanism of secretion, maturation and function of this model TPS protein.

Membrane Permeabilization by Bordetella Adenylate Cyclase Toxin Involves Pores of Tunable Size.

RTX (Repeats in ToXin) pore-forming toxins constitute an expanding family of exoproteins secreted by many Gram-negative bacteria and involved in infectious diseases caused by said pathogens. Despite the relevance in the host/pathogen interactions, the structure and characteristics of the lesions formed by these toxins remain enigmatic. Here, we capture the first direct nanoscale pictures of lytic pores formed by an RTX toxin, the Adenylate cyclase (ACT), secreted by the whooping cough bacterium Bordetella pertussis. We reveal that ACT associates into growing-size oligomers of variable stoichiometry and heterogeneous architecture (lines, arcs, and rings) that pierce the membrane, and that, depending on the incubation time and the toxin concentration, evolve into large enough "holes" so as to allow the flux of large molecular mass solutes, while vesicle integrity is preserved. We also resolve ACT assemblies of similar variable stoichiometry in the cell membrane of permeabilized target macrophages, proving that our model system recapitulates the process of ACT permeabilization in natural membranes. Based on our data we propose a non-concerted monomer insertion and sequential mechanism of toroidal pore formation by ACT. A size-tunable pore adds a new regulatory element to ACT-mediated cytotoxicity, with different pore sizes being putatively involved in different physiological scenarios or cell types.

Substrate specificity of the pyrophosphohydrolase LpxH determines the asymmetry of Bordetella pertussis lipid A.

Lipopolysaccharides are anchored to the outer membrane of Gram-negative bacteria by a hydrophobic moiety known as lipid A, which potently activates the host innate immune response. Lipid A of Bordetella pertussis, the causative agent of whooping cough, displays unusual structural asymmetry with respect to the length of the acyl chains at the 3 and 3' positions, which are 3OH-C10 and 3OH-C14 chains, respectively. Both chains are attached by the acyltransferase LpxA, the first enzyme in the lipid A biosynthesis pathway, which, in B. pertussis, has limited chain length specificity. However, this only partially explains the strict asymmetry of lipid A. In attempts to modulate the endotoxicity of B. pertussis lipid A, here we expressed the gene encoding LpxA from Neisseria meningitidis, which specifically attaches 3OH-C12 chains, in B. pertussis This expression was lethal, suggesting that one of the downstream enzymes in the lipid A biosynthesis pathway in B. pertussis cannot handle precursors with a 3OH-C12 chain. We considered that the UDP-diacylglucosamine pyrophosphohydrolase LpxH could be responsible for this defect as well as for the asymmetry of B. pertussis lipid A. Expression of meningococcal LpxH in B. pertussis indeed resulted in new symmetric lipid A species with 3OH-C10 or 3OH-C14 chains at both the 3 and 3' positions, as revealed by MS analysis. Furthermore, co-expression of meningococcal lpxH and lpxA resulted in viable cells that incorporated 3OH-C12 chains in B. pertussis lipid A. We conclude that the asymmetry of B. pertussis lipid A is determined by the acyl chain length specificity of LpxH.

Nucleobase Modified Adefovir (PMEA) Analogues as Potent and Selective Inhibitors of Adenylate Cyclases from Bordetella pertussis and Bacillus anthracis.

A series of 13 acyclic nucleoside phosphonates (ANPs) as bisamidate prodrugs was prepared. Five compounds were found to be non-cytotoxic and selective inhibitors of Bordetella pertussis adenylate cyclase toxin (ACT) in J774A.1 macrophage cell-based assays. The 8-aza-7-deazapurine derivative of adefovir (PMEA) was found to be the most potent ACT inhibitor in the series (IC50 =16 nm) with substantial selectivity over mammalian adenylate cyclases (mACs). AC inhibitory properties of the most potent analogues were confirmed by direct evaluation of the corresponding phosphonodiphosphates in cell-free assays and were found to be potent inhibitors of both ACT and edema factor (EF) from Bacillus anthracis (IC50 values ranging from 0.5 to 21 nm). Moreover, 7-halo-7-deazapurine analogues of PMEA were discovered to be potent and selective mammalian AC1 inhibitors (no inhibition of AC2 and AC5) with IC50 values ranging from 4.1 to 5.6 µm in HEK293 cell-based assays.

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

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 ß-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.

The importance of conserved amino acids in heme-based globin-coupled diguanylate cyclases.

Globin-coupled diguanylate cyclases contain globin, middle, and diguanylate cyclase domains that sense O2 to synthesize c-di-GMP and regulate bacterial motility, biofilm formation, and virulence. However, relatively few studies have extensively examined the roles of individual residues and domains of globin-coupled diguanylate cyclases, which can shed light on their signaling mechanisms and provide drug targets. Here, we report the critical residues of two globin-coupled diguanylate cyclases, EcGReg from Escherichia coli and BpeGReg from Bordetella pertussis, and show that their diguanylate cyclase activity requires an intact globin domain. In the distal heme pocket of the globin domain, residues Phe42, Tyr43, Ala68 (EcGReg)/Ser68 (BpeGReg), and Met69 are required to maintain full diguanylate cyclase activity. The highly conserved amino acids His223/His225 and Lys224/Lys226 in the middle domain of EcGReg/BpeGReg are essential to diguanylate cyclase activity. We also identified sixteen important residues (Leu300, Arg306, Asp333, Phe337, Lys338, Asn341, Asp342, Asp350, Leu353, Asp368, Arg372, Gly374, Gly375, Asp376, Glu377, and Phe378) in the active site and inhibitory site of the diguanylate cyclase domain of EcGReg. Moreover, BpeGReg266 (residues 1-266) and BpeGReg296 (residues 1-296), which only contain the globin and middle domains, can inhibit bacterial motility. Our findings suggest that the distal residues of the globin domain affect diguanylate cyclase activity and that BpeGReg may interact with other c-di-GMP-metabolizing proteins to form mixed signaling teams.

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.

Exposure to Bordetella pertussis adenylate cyclase toxin affects integrin-mediated adhesion and mechanics in alveolar epithelial cells.

BACKGROUND INFORMATION: The adenylate cyclase (CyaA) toxin is a major virulent factor of Bordetella pertussis, the causative agent of whooping cough. CyaA toxin is able to invade eukaryotic cells where it produces high levels of cyclic adenosine monophosphate (cAMP) affecting cellular physiology. Whether CyaA toxin can modulate cell matrix adhesion and mechanics of infected cells remains largely unknown. RESULTS: In this study, we use a recently proposed multiple bond force spectroscopy (MFS) with an atomic force microscope to assess the early phase of cell adhesion (maximal detachment and local rupture forces) and cell rigidity (Young's modulus) in alveolar epithelial cells (A549) for toxin exposure <1 h. At 30 min of exposure, CyaA toxin has a minimal effect on cell viability (>95%) at CyaA concentration of 0.5 nM, but a significant effect (≈81%) at 10 nM. MFS performed on A549 for three different concentrations (0.5, 5 and 10 nM) demonstrates that CyaA toxin significantly affects both cell adhesion (detachment forces are decreased) and cell mechanics (Young's modulus is increased). CyaA toxin (at 0.5 nM) assessed at three indentation/retraction speeds (2, 5 and 10 µm/s) significantly affects global detachment forces, local rupture events and Young modulus compared with control conditions, while an enzymatically inactive variant CyaAE5 has no effect. These results reveal the loading rate dependence of the multiple bonds newly formed between the cell and integrin-specific coated probe as well as the individual bond kinetics which are only slightly affected by the patho-physiological dose of CyaA toxin. Finally, theory of multiple bond force rupture enables us to deduce the bond number N which is reduced by a factor of 2 upon CyaA exposure (N ≈ 6 versus N ≈ 12 in control conditions). CONCLUSIONS: MFS measurements demonstrate that adhesion and mechanical properties of A549 are deeply affected by exposure to the CyaA toxin but not to an enzymatically inactive variant. This indicates that the alteration of cell mechanics triggered by CyaA is a consequence of the increase in intracellular cAMP in these target cells. SIGNIFICANCE: These results suggest that mechanical and adhesion properties of the cells appear as pertinent markers of cytotoxicity of CyaA toxin.