A activity of adenylate cyclase toxin PNAS PLUS mediates translocation of its adenylate cyclase domain

David González-Bullóna,b, Kepa B. Uribea,b, César Martína,b, and Helena Ostolazaa,b,1

aBiofisika Institute (UPV/EHU, CSIC), University of Basque Country, 48080 Bilbao, Spain; and bDepartment of Biochemistry and Molecular Biology, University of Basque Country (UPV/EHU), 48080 Bilbao, Spain

Edited by Pascale Cossart, Institut Pasteur, Paris, France, and approved June 30, 2017 (received for review February 6, 2017) Adenylate cyclase toxin (ACT or CyaA) plays a crucial role in can function independently as AC and hemolysin, respectively (14, respiratory tract colonization and virulence of the whooping cough 15). The hemolysin domain contains, in turn, a hydrophobic region causative bacterium Bordetella pertussis. Secreted as soluble pro- with amphipathic α-helices (∼500–750 residues) likely involved in tein, it targets myeloid cells expressing the CD11b/CD18 integrin pore-formation, two conserved Lys residues (Lys-863 and Lys-913) and on delivery of its N-terminal adenylate cyclase catalytic domain that are posttranslationally fatty acylated by a dedicated acyl- (AC domain) into the cytosol, generates uncontrolled toxic levels of (CyaC), and a C-terminal secretion signal recognized by cAMP that ablates bactericidal capacities of phagocytes. Our study a specific secretion machinery (CyaB, CyaD, and CyaE) (13, 16). + deciphers the fundamentals of the heretofore poorly understood ACT activity on cells requires binding of Ca2 ions into the nu- molecular mechanism by which the ACT domain directly merous (∼40) sites located in the C-terminal calcium-binding RTX crosses the host cell membrane. By combining molecular biology, repeats that are formed by Gly- andAsp-richnonapeptideshar- biochemistry, and biophysics techniques, we discover that ACT has boring a conserved X-(L/I/V)-X-G-G-X-G-X-D motif (17, 18). This intrinsic phospholipase A (PLA) activity, and that such activity deter- repeat domain has been reported to be involved in the toxin binding mines AC translocation. Moreover, we show that elimination of the to its specific cellular receptor, the CD11b/CD18 integrin (4, 19). ACT–PLA activity abrogates ACT toxicity in macrophages, particu- To generate cAMP into the host cytosol, ACT has to trans- larly at toxin concentrations close to biological reality of bacterial locate its catalytic domain from the hydrophilic extracellular infection. Our data support a molecular mechanism in which in situ medium into the hydrophobic environment of the membrane, – BIOCHEMISTRY generation of nonlamellar lysophospholipids by ACT PLA activity and then to the cell cytoplasm. How this is accomplished remains into the cell membrane would form, likely in combination with poorly understood. It is believed that after binding to the CD11b/ membrane-interacting ACT segments, a proteolipidic toroidal pore CD18 receptor, ACT inserts its amphipathic helices into the through which AC domain transfer could directly take place. Regu- plasma membrane and then delivers its catalytic domain directly – lation of ACT PLA activity thus emerges as novel target for thera- across the lipid bilayer into the cytosol (20, 21) without receptor- peutic control of the disease. mediated endocytosis (22). Temperatures above 15 °C (20), calcium at millimolar range, negative membrane potential, and bacterial toxins | RTX toxin family | protein translocation | biological unfolding of the AC domain (23) have been noted to be neces- membranes | membrane remodeling sary for AC delivery. Several studies have highlighted the importance of various he Gram-negative bacterium Bordetella pertussis causes amino acids and the contribution of distinct domains of the ACT Twhooping cough (pertussis), a highly contagious respiratory polypeptide in the translocation process (21, 24). However, a full disease that is an important cause of childhood morbidity and description at the molecular level of the individual steps that the mortality (1, 2). Despite widespread pertussis immunization in childhood, there are an estimated 50 million cases and 300,000 deaths Significance caused by pertussis globally each year. Infants who are too young to be vaccinated, children who are partially vaccinated, and fully vacci- nated persons with waning immunity are especially vulnerable to Numerous bacterial toxins can cross cell membranes, pene- disease (1). trating the cytosol of their target cells, but to do so exploits Bor- B. pertussis secretes several virulence factors, and among them cellular endocytosis or intracellular sorting machineries. detella pertussis adenylate cyclase toxin (ACT or CyaA) is crucial in the early steps adenylate cyclase toxin (ACT) delivers its cat- of colonization of the respiratory tract by the bacterium (3). Upon alytic domain directly across the cell membrane by an unknown mechanism, and generates cAMP, which subverts the cell sig- binding to the CD11b/CD18 integrin expressed on myeloid naling. Here, we decipher the fundamentals of the molecular phagocytes (4), ACT invades these immune cells by delivering into mechanism of ACT transport. We find that AC translocation their cytosol a calmodulin-activated adenylate cyclase (AC) do- and, consequently cytotoxicity, are determined by an intrinsic main that catalyzes the uncontrolled conversion of cytosolic ATP ACT–phospholipase A (PLA) activity, supporting a model in into cAMP (5), which causes inhibition of the oxidative burst and which in situ generation of nonlamellar lysophospholipids by complement-mediated opsonophagocytic killing of bacteria (6, 7). ACT–PLA activity remodels the cell membrane, forming pro- ACT also has hemolytic properties conferred by its C-terminal teolipidic toroidal “holes” through which AC domain transfer domain, which may form pores that permeabilize the cell mem- may directly take place. PLA-based specific protein transport in brane for monovalent cations, thus contributing to overall cytox- cells is unprecedented. icity of ACT toward phagocytes (8–10). Bordetella ACT toxin, together with the anthrax edema factor, are the two prototypes of Author contributions: D.G.-B., K.B.U., C.M., and H.O. designed research; D.G.-B. per- bacterial protein toxin that attack the immune response by over- formed research; D.G.-B., K.B.U., C.M., and H.O. analyzed data; and H.O. wrote the paper. boosting the major signaling pathway in the immune response The authors declare no conflict of interest. (cAMP, together with the MAPK pathway) (11). This article is a PNAS Direct Submission. ACT is a single polypeptide of 1,706 amino acids, constituted Freely available online through the PNAS open access option. by an AC enzyme domain (≈residues 1–400) and a C-terminal 1To whom correspondence should be addressed. Email: [email protected]. ≈ – hemolysin domain (residues 400 1,706) characteristic of the This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. RTX family of toxins to which ACT belongs (12, 13). Both activities 1073/pnas.1701783114/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1701783114 PNAS Early Edition | 1of10 Downloaded by guest on September 30, 2021 polypeptide follows to cross the membrane is still missing. De- acid (C16:0), and of a mass of 281.248 m/z (Fig. 1B), corresponding livery of the AC domain does not appear to proceed through the to free oleic acid (C18:1). Coincident with the appearance of free cation-selective pore formed by the hemolysin domain (25), but it fatty acids, there was a quantitative reduction (35% reduction) in the requires structural integrity of the hydrophobic segments (24), and relative abundance of the substrate with a mass of 760.586 m/z (Fig. it seems that the RTX hemolysin domain (residues 374–1,706) 1C) corresponding to intact POPC (C34:1). These data corroborate harbors all structural information required for AC translocation that ACT cleaves the sn-1 and sn-2 ester bonds in POPC, and thus (26). The region encompassing the helix 454–485 of ACT has been possesses PLA activity. recently noted as possibly playing an important role in promoting To identify more clearly whether the ACT–PLA activity translocation of the AC domain across the plasma membrane of cleaves specifically the sn-1 or the sn-2 ester bond of the lipid target cells (27, 28). A two-step model was proposed for AC substrate, we used highly sensitive fluorogenic phospholipid translocation: in a first step, and upon ACT binding to its CD11b/ substrates called PED-A1 [N-((6-(2,4-DNP)Amino)Hexanoyl)-1- CD18 integrin receptor, a translocation-competent ACT con- (BODIPY FL C5)-2-Hexyl-Sn-Glycero-3-Phosphoethanolamine] 2+ former would conduct extracellular Ca ions across the plasma (which measures PLA1 activity) and PED6 [N-((6-(2,4- membrane, which would activate a calpain-mediated cleavage of Dinitrophenyl)amino)hexanoyl)-2-(4,4-Difluoro-5,7-Dimethyl-4- talin, releasing the ACT–integrin complex from the cytoskeleton. Bora-3a,4a-Diaza-s-Indacene-3-Pentanoyl)-1-Hexadecanoyl-sn- – In a second step, this ACT integrin complex would move into Glycero-3-Phosphoethanolamine] (which measures PLA2 activity) lipid rafts, and there the cholesterol-rich lipid environment would (34). Both substrates are modified glycerophosphoethanolamines promote the translocation of the AC domain across the cell with BODIPY FL dye-labeled sn-1 or sn-2 acyl chains, respectively, membrane (29). A caveat of this model is the difficulty to explain and a dinitrophenyl group conjugated to the polar head group of how ACT translocates its AC domain into cells that do not express the lipid to provide intramolecular quenching. Thus, sn-1 or sn-2 the CD11b/CD18 integrin receptor (22, 30, 31). acyl chain cleavage by PLA1 or PLA2 activity eliminates the dye In recent years our laboratory has extensively explored ACT in- quenching, which results in increased fluorescence of the fluo- teraction with lipid membranes, and we have discovered that toxin rophore. As shown in Fig. 2, incubation of ACT (10 nM) with insertion into lipid bilayers induces transbilayer lipid motion (lipid DOPC liposomes containing PED-A1 substrate (20% molar ratio) “scrambling” or “flip-flop”) (32). Transbilayer lipid movement may induced an increase of the fluorescence measured at 515 nm, in- occur as a result of the insertion of foreign molecules (detergents, dicating the release of free fatty acid from the sn-1 ester bond of the lipids, or even proteins) in one of the membrane leaflets, or it may fluorescent substrate (Fig. 2A). Similarly, addition of ACT (10 nM) result from the enzymatic generation of lipids (e.g., lysophospholi- to DOPC liposomes containing PED6 (20% molar ratio) induced as pids, diacylglycerol, or ceramide) at one side of the membrane (33), well fluorescence increase at 515 nm (Fig. 2A), indicating that the and it has been involved in enhancing the transmembrane perme- sn-2 ester bond was also being hydrolyzed by ACT and free fatty ability of the lipid membranes (33). Here we sought to investigate acid released at the same time. Comparison of EC50 values whether ACT might possess intrinsic enzyme activity on lipids, obtained from the fluorescence data for both fluorescent substrates which could participate in AC domain translocation. We discovered at identical conditions (EC50 for PED-A1 = 2.42 nM; EC50 for that ACT is a calcium-dependent phospholipase A (PLA) that, by PED6 = 12.85 nM) indicated, however, that ACT cleaves with remodeling the cell membrane, mediates translocation of its cata- notably more efficiency the sn-1 ester bond of the fluorescent lytic domain in target macrophages. substrate than the sn-2 ester bond. For both fluorogenic substrates the fluorescence increase was proportional to the toxin concentra- Results tion added (Fig. 2B), and interestingly, the data indicated that the In Vitro ACT–PLA Activity. To explore potential enzyme activity of toxin PLA activity achieved saturation long before all of the sub- ACT on lipids, liposomes (LUV) composed of the common strate present was consumed (maximal relative fluorescence values membrane lipid phosphatidylcholine (PC) were incubated with for the hydrolysis of PED-A1 and PED6 substrates by the respective ACT (at four different lipid:protein molar ratios) for 30 min at positive controls, PLA1 and PLA2, at a concentration of 0.25 μg/mL 37 °C, after which we performed lipid extraction of the samples. upon incubation for 30 min at 37 °C, were 3.066 ± 0.046 and The extracted lipid fractions were spotted on silica plates together 2.896 ± 0.078, respectively). In conclusion, this set of experiments with appropriate lipid standards, and after being chromato- demonstrates that ACT displays PLA activity, with preference for graphed in one dimension (thin-layer chromatography, TLC) we the sn-1 ester bond, and hence, most likely that ACT acts as a performed quantification of the spots by densitometry. The spots phospholipase A1 (PLA1) on natural membranes. Importantly, it whose intensity was proportional to the lipid:toxin ratio used in also indicates that a “product inhibition” mechanism might operate each experiment were detected and found to migrate with an in ACT–PLA activity. Several proteins with PLA activities may also Rf value similar to the lipid standard corresponding to free fatty display lysoPLA activity (35); in contrast, ACT did not detectably acids, thus suggesting that ACT possess intrinsic PLA activity. hydrolyze either LysoPC (C18:1) or DLPC:LysoPC (C18:1) lipid Data from the densitometric analysis are depicted in Fig. S1.A substrates, suggesting that it is not a A (Fig. S2). control value, which was arbitrarily set as the unity, corresponds to To have an idea of the substrate specificity of the ACT–PLA the data obtained from liposomes treated without toxin. Because activity on several phospholipids, both neutral and anionic no spots corresponding to diacylglycerol, phosphatidic acid, or ceramide phospholipids were tested (Fig. S3). In this case, as a measure of were detected on the TLC gels analyzed, potential phospholipase the ACT–PLA activity, spots corresponding to free fatty acids C (PLC), (PLD), and sphingomyelinase activities were resolved by TLC and quantified by densitometry after in- were in principle discarded. cubation of ACT with liposomes made of different lipid sub- The ACT–PLA activity was further demonstrated by means of an strates (DOPC, DOPE, DMPG) at a lipid:protein ratio of ultrahigh performance liquid-chromatography mass spectrometry 1,000:1 for 30 min at 37 °C. Fig. S3 shows that ACT may hy- (UHPLC-MS) analysis of the products of an in vitro ACT phos- drolyze, in vitro, both neutral and anionic phospholipids with pholipase reaction (Fig. 1). POPC vesicles (1-palmitoyl-2-oleil- similar efficiency, indicating that ACT–PLA may not have a clear phosphatidylcholine) with distinct fatty acids, palmitic acid (C16:0) specificity for any given phospholipid. and oleic acid (C18:1) on sn-1 and sn-2 positions, respectively, were used as substrate. Superimposition of the chromatograms obtained ACT–PLA Harbors Conserved Ser/Asp Catalytic Sites, Sharing Similarity for the POPC substrate incubated with ACT showed a considerable with Patatin-Like . To characterize the ACT–PLA increase in the relative abundance of a mass of 255.233 m/z (Fig. activity in greater detail, analysis of the ACT amino acid sequence 1A), which corresponds to the reference standard for free palmitic was carried out with the multiple sequence alignment program

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Fig. 1. Identification of the reaction products of ACT-PLA activity by UHPLC-MSE. Identification of the reaction products of an in vitro PLA reaction on POPC lipid substrate were performed using UHPLC and simultaneous acquisition of exact mass at high and low collision energy, MSE. In the figure, representative − extracted ion UHPLC-ESI-MSE chromatograms are shown: electrospray ionization (ESI)−, m/z 255.233 of ion [Palmitic acid-H] (A), m/z 281.248 of ion [Oleic − + acid-H] (B), and ESI+, m/z 760.586 of ion [PC(16:0/18:1)+H] (C) obtained from samples containing POPC lipid substrate (green) or substrate exposed to ACT

(red). On top of the figure is a schematic representation of PLA1 and PLA2 cleavage sites on phospholipids is shown.

ClustalOmega (https://www.ebi.ac.uk), finding similarities with also inhibit with PLA1 activity (38). At a concentration several patatin-like phospholipases, such as the Pseudomonas of 10 nM, ACT was incubated with PED-A1–containing lipo- aeruginosa ExoU PLA2 (26.65% identity) or the Legionella pneu- somes (lipid:protein molar ratio 1,000:1) in the presence of in- mophila VipD PLA1 (21.60% identity) (Fig. S4). Patatin-like creasing amounts of MAFP (1–100 nM). Release of dye-labeled PLAs are most highly homologous to eukaryotic group IV cyto- sn-1 fatty acid from DOPC–PED-A1 liposomes by ACT–PLA solic PLA2α and group VI calcium-independent PLA2, which are activity was drastically halted by MAFP in a dose-dependent distinguished by a conserved Ser-Asp catalytic dyad within the manner (Fig. 3), which indicates that a catalytic Ser residue is , with catalytic Ser located in a conserved G-X-S-X-G involved in the ACT lipolytic activity. motif, and Asp in a D-X-G/A motif (boldface letters To further verify the involvement of the predicted catalytic correspond to conserved catalytic residues involved in PLA ac- residues in ACT–PLA activity, we constructed two ACT mutants, tivity) (36, 37). Remarkably, in its 1,706 amino acids long se- one in which Ser-606 was substituted by Ala (S606A), and a sec- quence, rich in Gly and Asp residues, ACT harbors a single G-X- ond mutant in which Asp-1079 was substituted by Ala (D1079A). S-X-G motif, with a GASAG sequence, which is localized in the A secondary structure of WT ACT and of the two mutant proteins predicted membrane-interacting α-helical region (residues 500– at 1-μM concentration (buffer with 10 mM CaCl2) was checked by 700) of ACT. On other side, the DXG/A motif that fits best in the far-UV circular dichroism (CD), and it was verified that the alignment analysis against the sequences of several patatin-like structure of the three proteins was very similar (Figs. S5 and S6). phospholipases has a DGG sequence, and it is localized in the PLA1 and PLA2 activities of the purified mutant proteins were + first one of the five Ca2 -binding β-rolls (block I, residues 1,014– checked using the substrates PED-A1 and PED6. As shown in Fig. 1,087) that form the so-called C-terminal repeat domain. Addi- 4, substitution of the Ser-606 by Ala had a dramatic effect on the tionally, along its C-terminal sequence ACT contains, in addition to toxin in vitro both PLA activities, so that the mutant protein the D1079GG motif, three more DXG motifs, but their fitting in the S606A was almost inactive in cleaving the fluorogenic substrates, sequence alignment assay was worse. relative to the intact ACT toxin. Substitution of Ser-606 by Ala did To ascertain whether ACT–PLA activity relies on the men- not affect any of the other ACT activities (production of cAMP in tioned Ser and Asp residues, and thus to experimentally validate solution, hemolytic activity) in the mutant protein ACT-S606A our prediction, we initially tested residue-specific agents, such as (Table S1). Replacement of Asp-1079 by Ala showed a less del- MAFP (methyl arachidonyl fluorophosphonate), for their ability eterious—although still substantial—decreasing effect on the mutant to inhibit ACT–PLA activity. MAFP is the MAFP analog of PLA1 activity, but its effect on the PLA2 activity was not significant arachidonic acid (AA), which is known to bind irreversibly to Ser (Fig. 4). Interestingly, this substitution also decreased the hemolytic residues and inhibit LysoPLA type I and PLA2 family members potency of the ACT-D1079A protein relative to WT ACT, whereas that exhibit LysoPLA activities (groups IV and VI) with an IC50 it did not alter at all of the cAMP-producing AC activity of the of 0.5–5 μM and other , such as fatty acid amide hy- mutant in solution (Table S1). Taken together, these results proved drolase, with an IC50 of 2.5–20 nM (38, 39). MAFP is known to that Ser-606 and Asp-1079 are directly involved in the ACT–PLA1

González-Bullón et al. PNAS Early Edition | 3of10 Downloaded by guest on September 30, 2021 Upon extensive cell washing, cells were then incubated with ACT (20 nM) for 10 min at 37 °C. After that incubation time, a solution of chloroform:methanol (1:1) was added to extract lipids, and then separation of free fatty acids from phospholipids (40) was carried out by TLC. Radioactivity in the sample extracted was quantified by scintillation counting. About 2- to 2.5-fold more 3H-arachidonic acid was detected in the samples exposed to ACT compared with control untreated cells (Fig. 5A), indicating that ACT exhibits PLA activity in vivo as well. As expected, the release of tritiated free AA from the J774A.1 cell membrane was notably reduced upon overnight preincubation of ACT with MAFP (Fig. 5A), thus corroborating the ACT–PLA activity features observed previously in the in vitro assays. Also as expected, the release of tritiated fatty acid was signifi- cantly reduced in cells exposed to the inactive S606A mutant (Fig. 5A). However, this inhibition by MAFP or mutation (S606A) observed in vivo was apparently less efficient than the observed in the in vitro assays (Figs. 3 and 4). We hypothesized that because eukaryotic cells contain endogenous PLAs, some of which are + Ca2 -dependent (41), it might be that part of the tritiated AA determined in our in vivo assays could come from the activity of such cellular PLAs. To settle the issue, we performed a control assay in which cells were preincubated with known inhibitors of both cytosolic and secretory cellular PLAs, MAFP and LY311727, respectively, prior to their treatment with ACT, ACT+MAFP, or ACT-S606A. Data from this experiment, depicted in Fig. 5B, confirmed our hypothesis and explained the apparent lower effi- cacy of inhibition. Fig. 5B shows, first, that as consequence of the inhibition of the cellular PLA2, the total amount of free arachi- donic acid quantified was inferior (≈1.7 times the control vs. 2.3 times the control), and second, that the inhibitory effect of MAFP or the point mutation ACT-S606A was now almost com- plete. In cells incubated with a lower ACT concentration (1.5 nM), the pattern observed was very similar, except that in this case the contribution of the intracellular phospholipases was inferior (Fig. S7). We can thus state that ACT exhibits PLA activity in vivo, releasing free fatty acids into the membrane. A striking finding here is that treatment of monocytes with ACT promotes acti- – Fig. 2. ACT PLA reaction determined by cleavage of PLA1-andPLA2-specific vation of cellular PLAs. We speculate that this activation might fluorogenic substrates. Two different specific fluorogenic phospholipid sub-

strates, PED-A1 and PED6, were used for measurement of PLA1 and PLA2 acti- vities, respectively. These substrates incorporate a BODIPY FL-dye–labeled sn-1 or a BODIPY FL-dye–labeled sn-2 acyl chain, and a dinitrophenyl quencher group.

Cleavage of the corresponding labeled-acyl chain by PLA1 or PLA2 activities results in the formation of nonfluorescent lysophospholipid and fluorescent fatty acid tagged to the fluorophore-BODIPY FL. The release of the corresponding fatty acid is determined as an increase of the fluorescence intensity at 515 nm (a.u.). The respective substrate hydrolysis was determined 30 min after in-

cubation of the substrate with the toxin (10 nM) at 37 °C. PLA1 and PLA2 ac- tivities are expressed as an increase of the fluorescence intensity at 515 nm (a.u.) relative to the corresponding control (lipid substrate in buffer, without toxin). The phospholipid substrates in the assay were liposomes (LUV) prepared with mixtures of DOPC:PED-A1 (4:1 mol:mol) or DOPC:PED6 (4:1 mol:mol) as described

in SI Materials and Methods (A). PLA1 and PLA2 activities expressed as increase of the fluorescence intensity at 515 nm (a.u.) as a function of ACT toxin concen- tration (0.5–40 nM) determined 30 min after incubation with the toxin (B). Data are expressed as average values ± SD from at least three independent experi- ments (n = 3).

catalytic reaction, corroborating our predictive data and suggesting that the catalytic mechanism of ACT–PLA might proceed through a serine-acyl intermediate, in the likeness of the patatin-like PLAs. Fig. 3. Dose-dependent inhibition of the ACT–PLA activity by MAFP, a residue specific irreversible PLA inhibitor. Release of dye-labeled sn-1 fatty – In Vivo ACT–PLA Activity. To investigate whether ACT exerts lipid acid from DOPC:PED-A1 (4:1 mol:mol) liposomes by ACT PLA1 activity was determined by fluorescence spectroscopy, in the presence of different con- acyl hydrolase activity in vivo, enzyme assays were next con- – ducted in J774A.1 macrophages exposed to the toxin. To this end, centrations of the PLA inhibitor MAFP, in the range 1 100 nM. PLA1 activity of ACT (10 nM) on DOPC:PED-A1 (4:1 mol:mol) liposomes after 30-min in- we first labeled the cell membranes with tritiated arachidonic acid cubation, normalized with respect to the control (substrate in buffer, 5 by an overnight incubation of the phagocytes (1 × 10 cells) in without toxin) was taken as 100% activity. Average values ± the SEM (n = 3) 3 6 medium with H-arachidonic acid (1.5 μCi/10 cells) at 37 °C. are shown. ***P < 0.001 with respect to intact ACT.

4of10 | www.pnas.org/cgi/doi/10.1073/pnas.1701783114 González-Bullón et al. Downloaded by guest on September 30, 2021 shown in Fig. 6A, preincubation of ACT with MAFP abrogated PNAS PLUS almost completely the accumulation of cAMP into the macro- phage cytosol. Possible side effects of the drug on ACT AC enzyme activity were ruled out, because cAMP production in solution by the MAFP-treated ACT was almost identical to in- tact ACT (Fig. 6B). Similarly, cAMP accumulation was impor- tantly halted in macrophages incubated with the S606A and D1079A mutants, whereas the point substitutions of the catalytic Ser and Asp did not have any effect on the capacity of the re- spective ACT mutants to generate cAMP in solution (Fig. 6 A and B). Therefore, these results proved that ACT is not capable of translocating efficiently its AC domain into the host cytosol if the toxin intrinsic PLA activity results abrogated, both chemi- cally (MAFP) or by mutagenesis. Thus, this set of experiments demonstrated a direct causal relationship between ACT–PLA activity and AC domain translocation. One uncontestable feature of the AC domain translocation process is the requirement of calcium in the millimolar range. To BIOCHEMISTRY

Fig. 4. ACT–PLA has Gly-X-Ser-X-Gly hydrolase motif. PLA1 (A)andPLA2 (B) activities of intact ACT, and of the ACT mutants S606A and D1079A, as a function of the corresponding protein concentration (0–40 nM), were determined

using the method of the PLA1-andPLA2-specific fluorogenic substrates PED- A1 and PED6. The S606A mutant has a point mutation in the amino acid Ser- 606 of the conserved Gly-X-Ser-X-Gly hydrolase motif, which was substituted by an Ala, and the D1079A mutant has a mutation in the Asp-1079 of the Asp-X-Gly/ Ala motif. Cleavage of the sn-1 and sn-2 ester bonds was followed as an increase of the fluorescence intensity at 515 nm determined 30 min after incubation with the corresponding protein toxin. Data are expressed as means ± SD (n = 3) from at least three independent experiments.

+ be mediated through ACT capacity to induce Ca2 elevations into target cells, which is greater the higher the toxin concentration is (42). Elucidation of the potential biological consequence of such activation in target cells needs further investigation.

ACT–PLA Activity Is Involved in AC Domain Translocation. To understand the biological consequence of the novel ACT phospholipase activ- ity, we hypothesized that it might modulate cellular processes re- lated to membrane remodeling. Lysophospholipids, one of the end products of the PLA activity, are nonlamellar lipids with intrinsic positive curvature (43), and formation of nonlamellar structures in the membrane is thought to be one way for “lipid pores” to occur in it (44). Moreover, insertion of amphipathic regions of many cellular proteins is now recognized as possibly resulting in the formation of hybrid proteolipidic “toroidal pores” by contributing to the fusion of Fig. 5. In vivo ACT–PLA activity. PLA activity of ACT was determined in inner and outer bilayer leaflets, in a way that is analogous to the macrophages by quantification of free [3H]-arachidonic acid released from effect on a pure lipid system of an external charge or the presence J774A.1 cells upon treatment with ACT for 10 min, as described in SI Mate- of detergents (45–47). rials and Methods.(A) PLA activity of intact ACT (20 nM), of ACT (20 nM) With this basis, we explored the possibility that ACT–PLA preincubated with the PLA inhibitor MAFP (25 μM), and of the ACT mutant activity could contribute in vivo to AC domain translocation by S606A (20 nM) was determined by the same procedure and graphically inducing membrane-remodeling effects through the generation depicted as the ratio of scintillation counting relative to control untreated of lysophospholipids into the cell membrane. As a measure of cells. (B) Cells were first preincubated with a combination of inhibitors of cytosolic and secreted PLA (MAFP + LY311727; each inhibitor at 25 μM) and the AC domain translocation efficiency, we quantified the in- then cells were treated with ACT, or ACT preincubated with MAFP (25 μM), tracellular cAMP accumulation induced by intact ACT (5 nM), or S606A proteins (20 nM), and upon 10-min incubation free [3H]-arachidonic by ACT preincubated with MAFP, and by the mutant proteins acid released was quantified. Average values ± the SEM (n = 3) are shown. S606A (5 nM) and D1079A (5 nM) in J774A.1 macrophages. As ***P < 0.001 with respect to intact ACT.

González-Bullón et al. PNAS Early Edition | 5of10 Downloaded by guest on September 30, 2021 determine whether the ACT–PLA activity was calcium-sensitive, the hydrolysis of the fluorogenic substrates was assayed in buffers with different CaCl2 concentrations, We found that the acyl 2+ hydrolase activity of ACT (PLA1 and PLA2) required Ca in the millimolar range (Fig. 6C), thus indicating that ACT is a calcium- dependent PLA. This was a key finding, because this dependence of the ACT–PLA activity for calcium may rationally explain at the molecular level the strict requirement shown by the AC translocation process for this divalent cation. Finally, we wanted to corroborate whether the mechanism by which the ACT–PLA activity dictates AC translocation is based on the membrane remodeling capacity of the lipid hydrolase action. With this aim, we determined the capacity of ACT to induce transbilayer lipid motion in DOPC vesicles, using for that a flip-flop assay previously adapted for this purpose in our laboratory (32). This assay is based on the phenomenon of FRET (32). In the present case, the donor and acceptor are, respectively, 7-nitrobenz-2-oxa-1,3- diazol-4-yl (NBD) and rhodamine. NBD is bound to a phospholipid initially located only in the inner monolayer of the membrane, and rhodamine is bound to a large protein (antibody) located outside the vesicle, so that at time 0 no energy transfer can occur. However, if transbilayer lipid motion occurs, some of the NBD lipid is trans- ferred to the outer monolayer and can interact with the external, protein-bound rhodamine, thus decreasing the fluorescence signal at 545 nm, the maximum of NBD emission. As expected, addition of ACT (10 nM) to DOPC liposomes induced a rapid reduction of the fluorescence signal at 545 nm, in a time scale of minutes (Fig. 7A), consistent with ACT capacity to promote lipid flip-flop, as previously noted by our group (32). In contrast, no changes in the fluorescence signal were detected upon addition of S606A or D1079A mutant proteins to liposomes (Fig. 7A), reflecting that the membrane restructuring capacity of ACT is directly linked to its intrinsic PLA activity. We provide a control of binding (Fig. S8)forthethree proteins used in the flip-flop assay, from which we ruled out that mutations on Ser-606 or Asp-1079 had gross effects on the ability of thoseproteinstobindtotheliposomemembranes. The concentration-dependent inhibitory effect of MAFP on the transbilayer lipid motion induced by ACT (Fig. 7B)wasafurther corroboration of the relation between ACT–PLA activity and membrane remodeling ability of ACT. This direct interrelation between both processes was furtherly proved into target cells, in which we could detect, through the widely used method of annexin V staining, that a few minutes (0–10 min) after the treatment of J774A.1 monocytes with ACT (5 nM), phosphatydylserine (PS; a phospholipid normally located in the cytoplasmic monolayer), was significantly located in the outer monolayer of the plasma mem- brane (Fig. 7C), and was reaffirmed by the experiments using the PLA inactive mutants ACT-S606A and ACT-D1079A, which were almost unable to promote such transmembrane lipid movement in the treated macrophages (Fig. 7C). Taken together, these results constitute a very solid indication that AC translocation relies on the generation of nonlamellar intermediates in the cell membrane by the ACT–PLA activity.

ACT–PLA Activity Is Instrumental to Induce Cell Cytotoxicity. Toxic action of ACT on phagocytes results primarily from the cytotoxic signaling of toxin-produced cAMP (12). Because production of cAMP into the host cell cytosol requires AC domain trans- Fig. 6. ACT–PLA activity is involved in AC domain translocation. The cata- location, we hypothesized that ablation of the ACT–PLA activity lytic activities of intact ACT, ACT preincubated with MAFP, and of the ACT would redound in a lower ACT cytotoxicity on macrophages. mutants S606A and D1079A, were measured in J774A.1 cells (A) or in solu- Therefore, we examined the effect of eliminating the ACT–PLA tion (B) at a toxin concentrations of 5 nM and 1 nM, respectively, and activity on the ACT-induced cytotoxicity, measured as lactate expressed as picomole cAMP generated per microgram of the corresponding dehydrogenase (LDH) release. If PLA activity is necessary for AC protein. The control sample corresponds to the cAMP amount measured in untreated cells. The cyclase activity was determined as described in SI Ma-

terials and Methods. Calcium-dependence of ACT–PLA1 activity was mea- sured for the three proteins, ACT, ACT-S606A, and ACT-D1079A, by the method of the fluorogenic substrate PED-A1, as a function of the calcium (C). Average values ± the SEM (n = 3) are shown. ***P < 0.001 with respect concentration, and expressed as relative fluorescence values (515 nm) in a.u. to intact ACT.

6of10 | www.pnas.org/cgi/doi/10.1073/pnas.1701783114 González-Bullón et al. Downloaded by guest on September 30, 2021 translocation, treatment of J774A.1 cells with the PLA-deficient PNAS PLUS S606A or D1079A ACT mutants would be expected to be less deleterious to cells. As shown in Fig. 8, intact ACT induced a progressive, concentration-dependent increase in cell cytotoxicity, inducing the release of LDH in about 70% of the cells after 2 h at a toxin concentration of 2 nM. In contrast, the PLA-deficient mutant ACT-S606A did not result in cytotoxicity at this same range of toxin concentrations (0–2 nM) (Fig. 8). Only at toxin concentrations above 2–4 nM did the cytoxicity induced by the PLA-deficient mutant ACT-S606A become substantial, very likely because of its intact pore-forming activity, which has been repor- ted that at this higher toxin concentrations synergizes with cAMP generation and ATP depletion in inducing cell death (9). The cytotoxicity induced by the ACT-D1079A mutant was almost negligable at both toxin concentration ranges (0–2nMand≥2nM), very likely because besides the PLA deficiency, this point mutation decreases the toxin hemolytic potency as well (Table S1), which contributes more importantly to general toxicity at the higher toxin concentrations (9). According to a recent study performed in a baboon model of infection (48), at the bacterium–target cell in- terface during infection of the respiratory tract, the concentration of ACT can be around 100 ng/mL (≈0.5–1 nM); thus, our results indicated that PLA activity is instrumental for ACT to be cytotoxic, particularly at toxin concentrations close to biological reality of bacterial infection. Discussion In this study, we decipher a fundamental event underlying the BIOCHEMISTRY hitherto elusive and unique mechanism of translocation of the B. pertussis ACT. We discover that ACT bears intrinsic PLA activity, both in vitro and in vivo. Our in vitro results obtained by means of two different biophysical techniques, highly sensitive fluorescent lipid substrates and MS, concur and clearly demonstrate that ACT hydrolyzes both the sn-1 and the sn-2 ester bonds of glycer- ophospholipid substrates, thus releasing into the membrane free fatty acids and lysophospholipids (Figs. 1 and 2). The compari- son of the efficiencies of the respective catalytic activities clearly indicates, however, that ACT cleaves with notably more effi- ciency the sn-1 ester bond of glycerophospholipid substrates, suggesting that ACT most likely acts as a PLA1 on natural membranes. Although many of the known PLA1 also display LysoPLA activities, we could not detect lysophospholipase ac- tivity in ACT (Fig. S2). Interestingly, our data also suggest that a “product inhibition” or “feedback-like” mechanism might oper- ate in the ACT–PLA activity (Fig. 2). This may be a relevant characteristic for the ACT–PLA mechanism, as we explain later, as it suggests that the ACT–PLA activity is not to massively “destroy” the target cell outer membrane lipids, but quite the opposite: that this ACT lipid hydrolytic activity has likely evolved as a “security mechanism” by inducing a local, subtle and “sur- gical” effect on the cell membrane of the target cells. The in vivo data obtained upon treatment of target macrophages with ACT (Fig. 5) are fully consistent with the in vitro results, and prove that ACT releases free fatty acids from the macrophage cell membrane in the same toxin concentration range in which it becomes cytotoxic for cells. Interestingly, we note here that ACT

different concentrations (50 nM and 1 μM) of the irreversible inhibitor MAFP Fig. 7. ACT induced membrane remodeling (lipid flip-flop) in liposomes and on the ACT flip-flop activity is shown. Lipid and protein concentrations were in macrophages. Time course of changes in fluorescence intensity of NBD as above (B). Externalization of the lipid PS to the outer monolayer of the represented as the ratio of the fluorescence intensities of the probe at 545/ cell membrane of macrophages, determined by flow cytometry as annexin V 575 nm, in control liposomes or upon incubation of LUVs with WT ACT or staining, by ACT, ACT-S606A, and ACT-D1079A. Macrophages were in- with the ACT–PLA inactive mutants S606A and D1079A. Lipid concentration cubated at 37 °C with the respective toxin at a concentration of 5 nM, for was 0.1 mM. ACT was added at 100 nM and liposomes were composed of 10 min. The control represents cells that were treated with medium without DOPC and NBD-PE (DOPC:NBD-PE, 1,000:6 molar ratio) (A). Effect of two toxin (C). Average values ± the SEM (n = 3) are shown.

González-Bullón et al. PNAS Early Edition | 7of10 Downloaded by guest on September 30, 2021 Of great significance in the ACT context is the elucidation here of the biological consequence of this novel ACT–PLA activity, that ACT lipid hydrolytic activity is directly linked to the AC domain transport across the plasma membrane of target phago- cytes. Two types of experiments allow us to conclude this. First, the sole inhibition of the ACT–PLA activity by MAFP prevents totally the accumulation of cAMP into the cytosol of the target cells, whereas it does not affect at all of the ACT production of cAMP in solution, thus proving that MAFP does not interfere with this second enzymatic activity of ACT (Fig. 6). Second, eradication of the ACT–PLA1 activity by introduction of a point substitution in the consensus catalytic center (S606, D1079) equally halts the accumulation of cAMP into the target cytosol (Fig. 6), whereas the mutated proteins remain fully functional in generation of cAMP in solution (Table S1). Hence, all of the results concur that AC transport across the membrane relies and is directly coupled to the hydrolysis of membrane phospholipids by ACT itself. Consistent with this essential role of the ACT–PLA activity in AC trans- location, we show that toxin-induced cell toxicity dramatically decreases in the macrophages treated with PLA-deficient ACT mutant proteins (Fig. 8), particularly at the toxin concentration- Fig. 8. Cell toxicity induced by low ACT concentrations requires toxin in- range close to biological reality of bacterial infection, indicating trinsic PLA activity. Cytotoxicity induced by WT ACT or by the ACT mutants that PLA activity is instrumental for ACT cytotoxicity. S606A and D1079A, in J774A.1 phagocytes after 2-h incubation at 37 °C, as a A key question here is how the ACT–PLA activity may mecha- function of toxin concentration is depicted. Cell cytotoxicity was determined nistically allow the transference of the AC domain across the through release of LDH into the culture medium, and is expressed as percent plasma membrane. The reaction products of ACT–PLA activity on LDH released into the medium relative to the total LDH content, after 2-h plasma membrane phospholipids are lysophospholipids and free incubation at 37 °C. Average values ± the SEM (n = 3) are shown. fatty acids. For decades it has been widely recognized that the modification of the cell-membrane composition through the direct + binding to target cells activates endogenous cellular Ca2 -sensi- action of hydrolytic enzymes, such as phospholipases on membrane tive PLAs (Fig. 5), most likely through its capacity to induce lipids, may regulate cell-membrane physical properties, such as the + Ca2 influx. At present, the biological consequences of such membrane mechanical stretching and bending constants (51), membrane curvature, or membrane lateral organization, favoring activation are unknown. – From the analysis of the toxin sequence, we conclude that the formation of transient nonlamellar structures (52 56). Lyso- phospholipids, nonlamellar lipids with positive spontaneous curva- ACT shares sequence similarity with members of the patatin-like ture, have been proven to induce formation of hydrophilic permeable phospholipases and mammalian cPLA (Fig. S4), suggesting that 2 proteolipidic “toroidal pores” in membranes in which amphipathic ACT may belong to the / hydrolase superfamily peptides and proteins are inserted, via a decrease in line tension (37). Consistent with this, we identify in the ACT toxin sequence 606 within the lipid bilayer because of relief of curvature stress along the twoconservedactive-sitecatalytic motifs, namely, the GAS AG 1079 membrane edge of the pore wall (44, 57). By definition, in a toroidal sequence (consensus motif GXSXG) and D GG sequence pore its constituent monolayers become continuous via the pore- (consensus motif DXG/A), and by means of pharmacological in- lining lipids (44, 57), and thus this structure allows the movement hibition and site-directed mutagenesis, we provide evidence of the of lipid molecules from one monolayer of the bilayer to the other. direct involvement of both Ser-606 and Asp-1079 as essential cat- Here we provide evidence that ACT–PLA activity is directly involved alytic residues in the ACT–PLA1 activity (Figs. 3–5), and thus we in promoting transbilayer lipid motion both in pure phosphatidyl- validate that ACT is a serine acylhydrolase. choline liposomes and, moreover, also in macrophages, and prove A first characterization of the substrate specificity of the that this is the direct inhibition of such ACT-induced lipid scrambling ACT–PLA activity shows that ACT may hydrolyze in vitro both (flip-flop) by MAFP and point mutations in the PLA catalytic sites neutral and anionic phospholipids (Fig. S3) with similar effi- (Fig. 7), thus indicating that ACT–PLA activity could form in the ciency, suggesting that ACT–PLA may not have a clear substrate lipid bilayer toroidal pores. Therefore, we provide herein an expla- specificity for any given phospholipid in the likeness of the nation to the question of how ACT–PLA activity may dictate AC patatin PLA enzyme (49). Recently, it has been shown that the domain transport through the plasma membrane. We postulate that – so-called ABH effector domain of the Vibrio cholerae MARTX in situ generation of nonlamellar lysophospholipids by ACT PLA toxin, another member of the RTX family of proteins to which activity on plasma membrane phospholipids would form a hydro- philic lipid pore of toroidal architecture through which AC domain ACT toxin belongs, possess PLA1 activity (50). Relative to the ACT–PLA activity, ABH appears to be highly specific for its transfer could directly take place. The location of the catalytic Ser- 606 within one of the predicted α-helices (helix IV) of the so-called phospholipid substrates, cleaving preferentially phosphatidyli- hydrophobic pore-forming domain of ACT, leads us to plausibly nositols, in particular PI3P (50). Compared with ACT however, hypothesize, that such a hydrophilic lipidic pore might be of pro- ABH takes several hours to hydrolyze PI3P (50), which might teolipidic nature, constituted likely by both lipids and one or more perhaps be because of an in vivo ABH requirement of some yet helical segments of the pore-forming region. Consistent with this – unknown intracellular . As shown here however, ACT idea, others have previously noted that mutations in specific amino PLA does not require any extracellular or intracellular cofactor acids of several α-helices from the hydrophobic domain impair not other than calcium to rapidly hydrolyze common membrane only the hemolytic activity, but also the translocation capacity of phospholipids; and given that ACT acts on the cell membrane ACT (21). outer monolayer where PC—in particular POPC and DOPC— Our translocation model is also consistent with the extraor- are the most abundant glycerophospholipids, we hypothesize dinary versatility of the AC domain to transport into the cytosol + that PC could be an in vivo likely ACT–PLA lipid substrate. of CD11b target cells large heterologous cargo polypeptides of

8of10 | www.pnas.org/cgi/doi/10.1073/pnas.1701783114 González-Bullón et al. Downloaded by guest on September 30, 2021 up to 200 residues in length within the AC domain (23, 26), as centrations, because at very low ACT concentrations (<100 ng/mL) PNAS PLUS among the unique characteristics of the proteolipidic toroidal at which AC translocation is supposed to be physiologically more pores are structural flexibility, low selectivity, and variable size relevant for cytotoxicity, the intracellular calcium elevation induced (44, 57). Direct participation in AC domain translocation of an by ACT is nearly null. Another feature also difficult to explain by ACT–PLA-generated hydrophilic toroidal tunnel, in which the Bumba et al.’s (29) model is how ACT can successfully transport, pore-walls may in part be lined by phospholipid head groups, upon fusion to the C-terminal ACT-hemolysin domain, such a va- may also be consistent with previous observations showing a net riety of N-terminal polypeptides carrying such different sequences positive charge requirement for AC domain to be translocated and lengths. A path that has to assist indistinctly the translocation of (58). Moreover, the possible formation of ion couples among a 400 amino acids-long chain or of other different polypeptides has those positively charged amino acid lateral chains with negatively to be expectedly dynamic, adaptable, and versatile, and these are charged phospholipid and fatty acid headgroups exposed at the typically features shared by lipid/protolipid toroidal pores, such as pore walls may represent an energy source that might contribute – to lowering the energy barrier for the AC domain insertion the ones that may be generated by a local PLA activity, as the ACT within the lipid bilayer (59, 60). PLA activity revealed here. Remarkably, our results may substantiate—at the molecular In conclusion, this study reveals intrinsic PLA activity of ACT level—previous data on calcium dependence of the ACT cata- that explains a fundamental molecular event underlying ACT lytic domain translocation in eukaryotic cells (20), and this is a protein translocation. The coupling in a single molecule of critical aspect that previous models on AC transport have failed membrane remodeling capacity—given by an enzymatic activity— to explain convincingly (20, 29). We show indeed that to be fully with the transport of proteins across membranes is unprecedented. operative, the ACT–PLA activity requires calcium in the milli- Because delivery of the AC domain is essential for ACT-induced molar range, thus providing a straightforward explanation to the cell toxicity, regulation of the ACT–PLA activity emerges as a strict calcium dependence of the AC transport across the cell novel drug target for the therapeutic control of whooping cough, an membrane (20). It is tempting to speculate that the requirement infectious disease that is the fifth largest cause of vaccine- of a cofactor, such as calcium ions for the ACT–PLA activity, preventable death in infants. might be linked to the absence of toxicity of ACT when expressed in bacteria, acting somehow as a security mechanism Materials and Methods for the prokaryotic cell. Expanded methods are available in SI Materials and Methods.

A model cited in the ACT field to explain AC translocation is the In vitro PLA activity was assayed using TLC and ulterior densitometric BIOCHEMISTRY two-step model proposed by Bumba et al. (29), in which in a first analysis, UHPLC-MS analysis, and selective fluorogenic phospholipid substrates: step, and upon ACT binding to its CD11b/CD18 integrin receptor, a namely, PED-A1 and PED6. For the in vivo PLA activity determination, cellular translocation-competent ACT conformer would conduct extracel- lipids were labeled with [3H]-arachidonic acid. Lipid flip-flop was assessed by + lular Ca2 ions across the plasma membrane, which it would acti- FRET using NBD-PE and rhodamine as donor and acceptor, respectively. vate a calpain-mediated cleavage of talin, releasing the ACT– integrin complex from the cytoskeleton. In a second step, this ACT– ACKNOWLEDGMENTS. We thank the technical and staff support provided integrin complex would move into lipid rafts, and there the by the Servicio de Lipidómica of the SGIker (Universidad del País Vasco/ cholesterol-rich lipid environment would promote the translocation Euskal Herriko Unibertsitatea, Ministeria de Ciencia e Innovación, GV/EJ, European Social Fund) and Rocío Alonso for excellent technical assistance. of the AC domain across the cell membrane (29). However, this This study was supported by Basque Government Grants Grupos Consolida- model cannot explain convincingly the uncontestable dependence dos IT849-13 and ETORTEK Program KK-2015/0000089. D.G.-B. and K.B.U. shown by the AC domain transport for millimolar calcium con- were recipients of a fellowship from the Bizkaia Biophysics Foundation.

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