Bordetella pertussis adenylate cyclase toxin. Structural and functional independence of the catalytic and hemolytic activities.

The Bordetella pertussis calmodulin-dependent adenylate cyclase (CyaA) is a 1706-residue-long toxin, endowed with hemolytic activity. We have constructed B. pertussis mutant strains producing modified CyaAs devoid of adenylate cyclase activity. Our results show that such modified CyaAs display hemolytic activity identical to the wild-type toxin, thus demonstrating that the hemolytic activity is independent of the adenylate cyclase activity. Furthermore, B. pertussis and Escherichia coli strains producing CyaA lacking the catalytic domain (residues 1-373) were constructed. The truncated protein exhibits hemolytic activity comparable to the wild-type toxin, thus establishing that the carboxyl-terminal 1332 residues alone are endowed with hemolytic activity. Together, these findings show that adenylate cyclase and hemolytic activities are located in two distinct regions of the molecule (respectively, approximately amino acids 1-400 and 401-1706) and that the two regions of CyaA are functionally independent.     

Mutation of specific acidic residues of the CNF1 T domain into lysine alters cell membrane translocation of the toxin

The Rho-GTPases-activating toxin CNF1 (cytotoxic necrotizing factor 1) delivers its catalytic activity into the cytosol of eukaryotic cells by a low pH membrane translocation mechanism reminiscent of that used by diphtheria toxin (DT). As DT, CNF1 exhibits a translocation domain (T) containing two predicted hydrophobic helices (H1-2) (aa 350-412) separated by a short peptidic loop (CNF1-TL) (aa 373-386) with acidic residues. In the DT loop, the loss of charge of acidic amino acids, as a result of protonation at low pH, is a critical step in the transfer of the DT catalytic activity into the cytosol. To determine whether the CNF1 T domain operates similarly to the DT T domain, we mutated several ionizable amino acids of CNF1-TL to lysine. Single substitutions such as D373K or D379K strongly decreased the cytotoxic effect of CNF1 on HEp-2 cells, whereas the double substitution D373K/D379K induced a nearly complete loss of cytotoxic activity. These single or double substitutions did not modify the cell-binding, enzymatic or endocytic activities of the mutant toxins. Unlike the wild-type toxin, single- or double-substituted CNF1 molecules bound to the HEp-2 plasma membrane could not translocate their enzymatic activity directly into the cytosol following a low pH pulse.     

Adenylate cyclase toxin from Bordetella pertussis. The relationship between induction of cAMP and hemolysis.

Bordetella pertussis produces a calmodulin-activated adenylate cyclase (AC) that exists in several forms. Only one form of AC, of apparent 200 kDa, is a toxin that penetrates eukaryotic cells and generates uncontrolled levels of intracellular cAMP. Recombination studies in transposon Tn5-insertion mutants of B. pertussis and amino acid sequence homology with alpha-hemolysin of Escherichia coli suggested that AC toxin may also have a hemolytic activity. Here, we demonstrate that only the toxic form of B. pertussis AC possesses hemolytic activity. Immunoblotting of membranes from sheep erythrocytes throughout the process of cell lysis detects the presence and accumulation of only the 200-kDa form of B. pertussis AC. cAMP generation induced by AC toxin in sheep erythrocytes is immediate whereas appearance of hemolysis is delayed by about 1 h and requires a higher level of AC toxin activity. Addition of exogenous calmodulin to sheep erythrocyte incubation medium potentiates the hemolytic activity of AC toxin but blocks cAMP generation. Extracellular Ca2+ at mM concentrations is absolutely required for cAMP generation but not for hemolysis. However, binding of AC toxin to sheep erythrocytes in the absence of exogenous Ca2+ followed by reincubation of cells in a toxin-free buffer containing Ca2+ leads to an immediate rise in intracellular cAMP. Human erythrocytes bind AC toxin and generate cAMP but are resistant to lysis. These results show that binding of AC toxin to erythrocytes can cause both cAMP generation and hemolysis or only one of these depending on conditions applied and cell type used.     

Relationships between heme incorporation, tetramer formation, and catalysis of a heme-regulated phosphodiesterase from Escherichia coli: a study of deletion and site-directed mutants

The heme-regulated phosphodiesterase (PDE) from Escherichia coli (Ec DOS) is a tetrameric protein composed of an N-terminal sensor domain (amino acids 1-201) containing two PAS domains (PAS-A, amino acids 21-84, and PAS-B, amino acids 144-201) and a C-terminal catalytic domain (amino acids 336-799). Heme is bound to the PAS-A domain, and the redox state of the heme iron regulates PDE activity. In our experiments, a H77A mutation and deletion of the PAS-B domain resulted in the loss of heme binding affinity to PAS-A. However, both mutant proteins were still tetrameric and more active than the full-length wild-type enzyme (140% activity compared with full-length wild type), suggesting that heme binding is not essential for catalysis. An N-terminal truncated mutant (DeltaN147, amino acids 148-807) containing no PAS-A domain or heme displayed 160% activity compared with full-length wild-type protein, confirming that the heme-bound PAS-A domain is not required for catalytic activity. An analysis of C-terminal truncated mutants led to mapping of the regions responsible for tetramer formation and revealed PDE activity in tetrameric proteins only. Mutations at a putative metal-ion binding site (His-590, His-594) totally abolished PDE activity, suggesting that binding of Mg2+ to the site is essential for catalysis. Interestingly, the addition of the isolated PAS-A domain in the Fe2+ form to the full-length wild-type protein markedly enhanced PDE activity (>5-fold). This activation is probably because of structural changes in the catalytic site as a result of interactions between the isolated PAS-A domain and that of the holoenzyme.     

A receptor-binding region in Escherichia coli alpha-haemolysin

Escherichia coli alpha-hemolysin (HlyA) is a 107-kDa protein toxin with a wide range of mammalian target cells. Previous work has shown that glycophorin is a specific receptor for HlyA in red blood cells (Cortajarena, A. L., Go?i, F. M., and Ostolaza, H. (2001) J. Biol. Chem. 276, 12513-12519). The present study was aimed at identifying the glycophorin-binding region in the toxin. Data in the literature pointed to a short amino acid sequence near the C terminus as a putative receptor-binding domain. Previous sequence analyses of several homologous toxins that belong, like HlyA, to the so-called RTX toxin family revealed a conserved region that corresponded to residues 914-936 of HlyA. We therefore prepared a deletion mutant lacking these residues (HlyA Delta 914-936) and found that its hemolytic activity was decreased by 10,000-fold with respect to the wild type. This deletion mutant was virtually unable to bind human and horse red blood cells or to bind pure glycophorin in an affinity column. The peptide Trp914-Arg936 had no lytic activity of its own, but it could bind glycophorin reconstituted in lipid vesicles. Moreover, the peptide Trp914-Arg936 protected red blood cells from hemolysis induced by wild type HlyA. It was concluded that amino acid residues 914-936 constitute a major receptor-binding region in alpha-hemolysin.     

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.     

Autoprocessing of the Vibrio cholerae RTX toxin by the cysteine protease domain

Vibrio cholerae RTX is a large multifunctional bacterial toxin that causes actin crosslinking. Due to its size, it was predicted to undergo proteolytic cleavage during translocation into host cells to deliver activity domains to the cytosol. In this study, we identified a domain within the RTX toxin that is conserved in large clostridial glucosylating toxins TcdB, TcdA, TcnA, and TcsL; putative toxins from V. vulnificus, Yersinia sp., Photorhabdus sp., and Xenorhabdus sp.; and a filamentous/hemagglutinin-like protein FhaL from Bordetella sp. In vivo transfection studies and in vitro characterization of purified recombinant protein revealed that this domain from the V. cholerae RTX toxin is an autoprocessing cysteine protease whose activity is stimulated by the intracellular environment. A cysteine point mutation within the RTX holotoxin attenuated actin crosslinking activity suggesting that processing of the toxin is an important step in toxin translocation. Overall, we have uncovered a new mechanism by which large bacterial toxins and proteins deliver catalytic activities to the eukaryotic cell cytosol by autoprocessing after translocation.     

Putative identification of an amphipathic alpha-helical sequence in hemolysin of Escherichia coli (HlyA) involved in transmembrane pore formation

Abstract Escherichia coli hemolysin is a pore-forming protein belonging to the RTX toxin family. Cysteine scanning mutagenesis was performed to characterize the putative pore-forming domain of the molecule. A single cysteine residue was introduced at 48 positions within the sequence spanning residues 170-400 and labeled with the polarity-sensitive dye badan. Spectrofluorimetric analyses indicated that several amino acids in this domain are inserted into the lipid bilayer during pore formation. An amphipathic alpha-helix spanning residues 272-298 was identified that may line the aqueous pore. The importance of this sequence was highlighted by the introduction of two prolines at positions 284 and 287. Disruption of the helix structure did not affect binding properties, but totally abolished the hemolytic activity of the molecule.     

Quantitative method for determination of hemolytic activity of Clostridium septicum toxin

A quantitative method for titration of hemolytic activity of Clostridium septicum toxin was established. No linear dose-response line was obtained between the toxin dose and hemolysis ratio. Logit or Probit transformation of the hemolysis ratio changed this log-dose response curve into a linear line. We recommend a highly reproducible and accurate method based on parallel line assay using a well-standardized reference toxin for determination of hemolytic activity. The method consists of simultaneous titrations of the hemolysis ratios with each sample and a reference toxin, Logit or Probit transformation of the hemolysis ratio, and expression of the activity in the relative hemolytic value.     

Cloning and characterization of two bistructural S-layer-RTX proteins from Campylobacter rectus

Campylobacter rectus is an important periodontal pathogen in humans. A surface-layer (S-layer) protein and a cytotoxic activity have been characterized and are thought to be its major virulence factors. The cytotoxic activity was suggested to be due to a pore-forming protein toxin belonging to the RTX (repeats in the structural toxins) family. In the present work, two closely related genes, csxA and csxB (for C. rectus S-layer and RTX protein) were cloned from C. rectus and characterized. The Csx proteins appear to be bifunctional and possess two structurally different domains. The N-terminal part shows similarity with S-layer protein, especially SapA and SapB of C. fetus and Crs of C. rectus. The C-terminal part comprising most of CsxA and CsxB is a domain with 48 and 59 glycine-rich canonical nonapeptide repeats, respectively, arranged in three blocks. Purified recombinant Csx peptides bind Ca2+. These are characteristic traits of RTX toxin proteins. The S-layer and RTX domains of Csx are separated by a proline-rich stretch of 48 amino acids. All C. rectus isolates studied contained copies of either the csxA or csxB gene or both; csx genes were absent from all other Campylobacter and Helicobacter species examined. Serum of a patient with acute gingivitis showed a strong reaction to recombinant Csx protein on immunoblots.     

Characterization of functional domains of the SMN protein in vivo

The Survival of Motor Neurons (SMN) is the disease gene of spinal muscular atrophy. We have previously established a genetic system based on the chicken pre-B cell line DT40, in which expression of SMN protein is regulated by tetracycline, to study the function of SMN in vivo. Depletion of SMN protein is lethal to these cells. Here we tested the functionality of mutant SMN proteins by determining their capacity to rescue the cells after depletion of wild-type SMN. Surprisingly, all of the spinal muscular atrophy-associated missense mutations tested were able to support cell viability and proliferation. Deletion of the amino acids encoded by exon 7 of the SMN gene resulted in a partial loss of function. A mutant SMN protein lacking both the tyrosine/glycine repeat (in exon 6) and exon 7 failed to sustain viability, indicating that the C terminus of the protein is critical for SMN activity. Interestingly, the Tudor domain of SMN, encoded by exon 3, does not appear to be essential for SMN function since a mutant deleted of this domain restored cell viability. Unexpectedly, a chicken SMN mutant (DeltaN39) lacking the N-terminal 39 amino acids that encompass the Gemin2-binding domain also rescued the lethal phenotype. Moreover, the level of Gemin2 in DeltaN39-rescued cells was significantly reduced, indicating that Gemin2 is not required for DeltaN39 to perform the essential function of SMN in DT40 cells. These findings suggest that SMN may perform a novel function in DT40 cells.     

Recognition of sphingomyelin by lysenin and lysenin-related proteins

Lysenin is a sphingomyelin (SM)-specific toxin isolated from the coelomic fluid of the earthworm Eisenia foetida. Lysenin comprises a family of proteins together with lysenin-related protein 1 (LRP-1, lysenin 2) and LRP-2 (lysenin 3). In the present study, we characterized LRP-1 and LRP-2 together with lysenin using maltose-binding-protein-tagged recombinant proteins. LRP-2 specifically bound SM and induced hemolysis like lysenin. In contrast the binding and hemolytic activities of LRP-1 were 10 times less than those of lysenin and LRP-2. Lysenin and LRP-2 share 30 common sites of aromatic amino acids. Among them, only one position, phenylalanine 210, is substituted for isoleucine in LRP-1. The activity of LRP-1 was dramatically increased by introducing a single amino acid substitution of isoleucine 210 to phenylalanine, suggesting the importance of this aromatic amino acid in biological activities of lysenin and LRPs. The importance of aromatic amino acids was further indicated by a systematic tryptophan to alanine mutation of lysenin. Lysenin contains six tryptophan residues of which five are conserved in LRP-1 and -2. We showed that the conserved tryptophans but not the nonconserved one were required both in the recognition of SM and in the hemolytic activity of lysenin. Our results suggest the importance of tryptophan in the toxin function likely due to a direct recognition of SM or in maintaining the protein structure.     

Identification of a direct hemolytic effect dependent on the catalytic activity induced by phospholipase‐D (dermonecrotic toxin) from brown spider venom

Brown spiders have world‐wide distribution and are the cause of health problems known as loxoscelism. Necrotic cutaneous lesions surrounding the bites and less intense systemic signs like renal failure, DIC, and hemolysis were observed. We studied molecular mechanism by which recombinant toxin, biochemically characterized as phospholipase‐D , causes direct hemolysis (complement independent). Human erythrocytes treated with toxin showed direct hemolysis in a dose‐dependent and time‐dependent manner, as well as morphological changes in cell size and shape. Erythrocytes from human, rabbit, and sheep were more susceptible than those from horse. Hemolysis was not dependent on ABO group or Rhesus system. Confocal and FACS analyses using antibodies or GFP‐phospholipase‐D protein showed direct toxin binding to erythrocytes membrane. Moreover, toxin‐treated erythrocytes reacted with annexin‐V and showed alterations in their lipid raft profile. Divalent ion chelators significantly inhibited hemolysis evoked by phospholipase‐D , which has magnesium at the catalytic domain. Chelators were more effective than PMSF (serine‐protease inhibitor) that had no effect on hemolysis. By site‐directed mutation at catalytic domain (histidine 12 by alanine), hemolysis and morphologic changes of erythrocytes (but not the toxin's ability of membrane binding) were inhibited, supporting that catalytic activity is involved in hemolysis and cellular alterations but not toxin cell binding. The results provide evidence that L. intermedia venom phospholipase‐D triggers direct human blood cell hemolysis in a catalytic‐dependent manner. J. Cell. Biochem. 107: 655–666, 2009. © 2009 Wiley‐Liss, Inc.     

Biologically active peptides of the vesicular stomatitis virus glycoprotein.

A peptide corresponding to the amino-terminal 25 amino acids of the mature vesicular stomatitis virus glycoprotein has recently been shown to be a pH-dependent hemolysin. In the present study, we analyzed smaller constituent peptides and found that the hemolytic domain resides within the six amino-terminal amino acids. Synthesis of variant peptides indicates that the amino-terminal lysine can be replaced by another positively charged amino acid (arginine) but that substitution with glutamic acid results in the total loss of the hemolytic function. Peptide-induced hemolysis was dependent upon buffer conditions and was inhibited when isotonicity was maintained with mannitol, sucrose, or raffinose. In sucrose, all hemolytic peptides were also observed to mediate hemagglutination. The large 25-amino acid peptide is also a pH-dependent cytotoxin for mammalian cells and appears to effect gross changes in cell permeability. Conservation of the amino terminus of vesicular stomatitis virus and rabies virus suggests that the membrane-destabilizing properties of this domain may be important for glycoprotein function.     

An amphipathic alpha-helix including glutamates 509 and 516 is crucial for membrane translocation of adenylate cyclase toxin and modulates formation and cation selectivity of its membrane channels

The Bordetella pertussis adenylate cyclase toxin-hemolysin (ACT or CyaA) is a multifunctional protein. It forms small cation-selective channels in target cell and lipid bilayer membranes and it delivers into cell cytosol the amino-terminal adenylate cyclase (AC) domain, which catalyzes uncontrolled conversion of ATP to cAMP and causes cell intoxication. Here, we demonstrate that membrane translocation of the AC domain into cells is selectively dissociated from ACT membrane insertion and channel formation when a helix-breaking proline residue is substituted for glutamate 509 (Glu-509) within a predicted transmembrane amphipathic alpha-helix. Neutral substitutions of Glu-509 had little effect on toxin activities. In contrast, charge reversal by lysine substitutions of the Glu-509 or of the adjacent Glu-516 residue reduced the capacity of the toxin to translocate the AC domain across membrane and enhanced significantly its specific hemolytic activity and channel forming capacity in lipid bilayer membranes. Combination of the E509K and E516K mutations in a single molecule further exacerbated hemolytic and channel forming activity and ablated translocation of the AC domain into cells. The lysine substitutions strongly decreased the cation selectivity of the channels, indicating that Glu-509 and Glu-516 are located within or close to the membrane channel. These results suggest that the structure including glutamate residues 509 and 516 is critical for AC membrane translocation and channel forming activity of ACT.     

Structural and functional characterization of an essential RTX subdomain of Bordetella pertussis adenylate cyclase toxin

The adenylate cyclase toxin (CyaA) is one of the major virulence factors of Bordetella pertussis, the causative agent of whooping cough. CyaA is able to invade eukaryotic cells by a unique mechanism that consists in a calcium-dependent, direct translocation of the CyaA catalytic domain across the plasma membrane of the target cells. CyaA possesses a series of a glycine- and aspartate-rich nonapeptide repeats (residues 1006-1613) of the prototype GGXG(N/D)DX(L/I/F)X (where X represents any amino acid) that are characteristic of the RTX (repeat in toxin) family of bacterial cytolysins. These repeats are arranged in a tandem fashion and may fold into a characteristic parallel beta-helix or beta-roll motif that constitutes a novel type of calcium binding structure, as revealed by the three-dimensional structure of the Pseudomonas aeruginosa alkaline protease. Here we have characterized the structure-function relationships of various fragments from the CyaA RTX subdomain. Our results indicate that the RTX functional unit includes both the tandem repeated nonapeptide motifs and the adjacent polypeptide segments, which are essential for the folding and calcium responsiveness of the RTX module. Upon calcium binding to the RTX repeats, a conformational rearrangement of the adjacent non-RTX sequences may act as a critical molecular switch to trigger the CyaA entry into target cells.     

Structure-function studies of the adenylate cyclase toxin of Bordetella pertussis and the leukotoxin of Pasteurella haemolytica by heterologous C protein activation and construction of hybrid proteins.

The adenylate cyclase toxin (CyaA) from Bordetella pertussis and the leukotoxin (LktA) from Pasteurella haemolytica are members of the RTX (stands for repeats in toxin) family of cytolytic toxins. They have pore-forming activity and share significant amino acid homology but show marked differences in biological activity. CyaA is an invasive adenylate cyclase and a weak hemolysin which is active on a wide range of mammalian cells. LktA is a cytolytic protein with a high target cell specificity and is able to lyse only leukocytes and platelets from ruminants. Each toxin is synthesized as an inactive protoxin encoded by the A gene, and the product of the accessory C gene is required for posttranslational activation. Heterologous activation of LktA by CyaC did not result in a change in its specificity for nucleated cells, although the toxin showed a greater hemolytic-to-cytotoxic ratio. LktC was unable to activate CyaA. A hybrid toxin (Hyb1), which contained the N-terminal enzymic domain and the pore-forming domain from CyaA (amino acids [aa] 1 to 687), with the remainder of the protein derived from the C-terminal end of LktA (aa 379 to 953), showed no toxic activity. Replacement of part of the LktA C-terminal domain of Hyb1 by the CyaA C-terminal domain (aa 919 to 1706) to create hybrid toxin 2 (Hyb2) partially restored toxic activity. In contrast to CyaA, Hyb2 was activated more efficiently by LktC than by CyaC, showing the importance of the region between aa 379 and 616 of LktA for activation by LktC. LktC-activated Hyb2 was more active against ruminant than murine nucleated cells, whereas CyaC-activated Hyb2 displayed a similar, but lower, activity against both cell types. These data indicate that LktC and the region with which it interacts have an influence on the target cell specificity of the mature toxin.     

Role of tryptophan-1 in hemolytic and phospholipase C activities of Clostridium perfringens alpha-toxin

Replacement of the Trp-1 in Clostridium perfringens alpha-toxin with tyrosine caused no effect on hemolytic and phospholipase C (PLC) activities or on binding to the zinc ion, but that of the residue with alanine, glycine and histidine led to drastic decreases in these activities and a significant reduction in binding to the zinc ion. The hemolytic and PLC activities of W1H and W1A were significantly increased by the preincubation of these variant toxins with zinc ions, but the preincubation of W1G with the metal ion caused little effect on these activities. Gly-Ile-alpha-toxin, which contained an additional Gly-Ile linked to the N-terminal amino acid of alpha-toxin, did not show hemolytic activity, but showed about 6% PLC activity of the wild-type toxin. A mutant toxin, which contained an additional Gly-Ile linked to the N-terminus of a protein lacking 4 N-terminal residues of alpha-toxin, showed about 1 and 6% hemolytic and PLC activities of the wild-type toxin, respectively. Incubation of the mutant toxin with zinc ions caused a significant increase in PLC activity. These observations suggested that Trp-1 is not essential for toxin activity, but plays a role in binding to zinc ions.     

Characterization of a Membrane-active Peptide from the Bordetella pertussis CyaA Toxin

Bordetella pertussis, the pathogenic bacteria responsible for whooping cough, secretes several virulence factors, among which is the adenylate cyclase toxin (CyaA) that plays a crucial role in the early stages of human respiratory tract colonization. CyaA invades target cells by translocating its catalytic domain directly across the plasma membrane and overproduces cAMP, leading to cell death. The molecular process leading to the translocation of the catalytic domain remains largely unknown. We have previously shown that the catalytic domain per se, AC384, encompassing residues 1–384 of CyaA, did not interact with lipid bilayer, whereas a longer polypeptide, AC489, spanning residues 1–489, binds to membranes and permeabilizes vesicles. Moreover, deletion of residues 375–485 within CyaA abrogated the translocation of the catalytic domain into target cells. Here, we further identified within this region a peptidic segment that exhibits membrane interaction properties. A synthetic peptide, P454, corresponding to this sequence (residues 454–485 of CyaA) was characterized by various biophysical approaches. We found that P454 (i) binds to membranes containing anionic lipids, (ii) adopts an α-helical structure oriented in plane with respect to the lipid bilayer, and (iii) permeabilizes vesicles. We propose that the region encompassing the helix 454–485 of CyaA may insert into target cell membrane and induce a local destabilization of the lipid bilayer, thus favoring the translocation of the catalytic domain across the plasma membrane.