Repeats In Toxin Research Articles

Teneurin-1, a vertebrate homologue of the Drosophila pair-rule gene ten-m, is a neuronal protein with a novel type of heparin-binding domain.

The Drosophila gene ten-m is the first pair-rule gene not encoding a transcription factor, but an extracellular protein. We have characterized a highly conserved chicken homologue that we call teneurin-1. The C-terminal part harbors 26 repetitive sequence motifs termed YD-repeats. The YD-repeats are most similar to the core of the rhs elements of Escherichia coli. Related repeats in toxin A of Clostridium difficile are known to bind specific carbohydrates. We show that recombinantly expressed proteins containing the YD-repeats of teneurin-1 bind to heparin. Furthermore, heparin lyase treatment of extracts of cells expressing recombinant YD-repeat protein releases this protein from high molecular mass aggregates. In situ hybridization and immunostaining reveals teneurin-1 expression in neurons of the developing visual system of chicken and Drosophila. This phylogenetic conservation of neuronal expression from flies to birds implies fundamental roles for teneurin-1 in neurogenesis. This is supported by the neurite outgrowth occurring on substrates made of recombinant YD-repeat proteins, which can be inhibited by heparin. Database searches resulted in the identification of ESTs encoding at least three further members of the teneurin family of proteins. Furthermore, the human teneurin-1 gene could be identified on chromosome Xq24/25, a region implied in an X-linked mental retardation syndrome.

HlyC, the internal protein acyltransferase that activates hemolysin toxin: the role of conserved tyrosine and arginine residues in enzymatic activity as probed by chemical modification and site-directed mutagenesis.

Internal fatty acylation of proteins is a recognized means of modifying biological behavior. Escherichia coli hemolysin A (HlyA), a toxic protein, is transcribed as a nontoxic protein and made toxic by internal acylation of two lysine residue epsilon-amino groups; HlyC catalyzes the acyl transfer from acyl-acyl carrier protein (ACP), the obligate acyl donor. Conserved residues among the respective homologous C proteins that activate 13 different RTX (repeats in toxin) toxins of which HlyA is the prototype likely include some residues that are important in catalysis. Possible roles of two conserved tyrosines and two conserved arginines were investigated by noting the effects of chemical modifiers and site-directed mutagenesis. TNM modification of HlyC at pH 8.0 led to extensive inhibition that was prevented by the presence of the substrate myristoyl-ACP but not by the product, ACPSH. NAI had no effect. Y70G and Y150G greatly diminished enzyme activity, whereas mutations Y70F and Y150F exhibited wild-type activity. Modification of arginine residues with PG markedly lowered acyltransferase activity with moderate protection by both myristoyl-ACP and ACPSH. Under optimum conditions, four separate mutations of the two conserved arginine residues (R24A, R24K, R87A, and R87K) had little effect on acyltransferase activity.

Lipopolysaccharide complexes with Pasteurella haemolytica leukotoxin.

The presence of lipopolysaccharide (LPS) in gram-negative bacterial repeats-in-toxin (RTX) toxin preparations, as well as the harsh conditions required to remove it, suggests that LPS may complex with RTX toxins. Concentrated culture supernatant (CCS) preparations of the RTX toxin Pasteurella haemolytica leukotoxin (LKT) contained LKT and LPS as the most prominent components, with LKT and LPS constituting approximately 30 and 50% of the density of the silver-stained fraction on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), respectively. CCS LKT contained 3.69 +/- 0.46 mg of LPS per mg of protein, which was estimated to indicate an LPS/LKT molar ratio of approximately 60:1. Subjection of the CCS LKT to preparative SDS-PAGE resulted in separation of LPS from LKT as detected by silver-stained analytical SDS-PAGE; however, the LKT fraction (SDS-PAGE LKT) contained significant endotoxin activity as detected by the Limulus amebocyte lysate assay. Subjection of the SDS-PAGE LKT to a second preparative SDS-PAGE run resulted in a reduction of the LPS/LKT molar ratio to 1:20. The target cell specificity of LKT for bovine leukocytic cells was retained by the SDS-PAGE LKT, and isolated LPS at comparable concentrations to that in CCS LKT exhibited no leukolytic activity. Addition of isolated LPS back to SDS-PAGE LKT resulted in reconstitution of an LPS-LKT complex. Immediately following reconstitution of the LPS-LKT complex, there was minimal change in leukolytic activity of the complex, but following 9.5 h at temperatures from -135 to 37 degrees C, the LPS-LKT complex exhibited increased leukolytic activity and thermal stability compared to SDS-PAGE LKT. Therefore, it appears that LPS complexes with LKT, resulting in enhanced and stabilized leukolytic activity.

Epitope mapping of monoclonal antibodies against Bordetella pertussis adenylate cyclase toxin.

Adenylate cyclase (AC) toxin from Bordetella pertussis is a 177-kDa repeats-in-toxin (RTX) family protein that consists of four principal domains; the catalytic domain, the hydrophobic domain, the glycine/aspartate-rich repeat domain, and the secretion signal domain. Epitope mapping of 12 monoclonal antibodies (MAbs) directed against AC toxin was conducted to identify regions important for the functional activities of this toxin. A previously developed panel of in-frame deletion mutants of AC toxin was used to localize MAb-specific epitopes on the toxin. The epitopes of these 12 MAbs were located throughout the toxin molecule, recognizing all major domains. Two MAbs recognized a single epitope on the distal portion of the catalytic domain, two reacted with the C-terminal 217 amino acids, one bound to the hydrophobic domain, and one bound to either the hydrophobic domain or the functionally unidentified region adjacent to it. The remaining six MAbs recognized the glycine/aspartate-rich repeat region. To localize these six MAbs, different peptides derived from the repeat region were constructed. Two of the six MAbs appeared to react with the repetitive motif and exhibited cross-reactivity with Escherichia coli hemolysin. The remaining four MAbs appeared to interact with unique epitopes within the repeat region. To evaluate the roles of these epitopes on toxin function, each MAb was screened for its effect on intoxication (cyclic AMP accumulation) and hemolytic activity. The two MAbs recognizing the distal portion of the catalytic domain blocked intoxication of Jurkat cells by AC toxin but had no effect on hemolysis. On the other hand, a MAb directed against a portion of the repeat region caused partial inhibition of AC toxin-induced hemolysis without affecting intoxication. In addition, the MAb recognizing either the hydrophobic domain or the unidentified region adjacent to it inhibited both intoxication and hemolytic activity of AC toxin. These findings extend our understanding of the regions necessary for the complex events required for the biological activities of AC toxin and provide a set of reagents for further study of this novel virulence factor.

Identification of a vibrio cholerae RTX toxin gene cluster that is tightly linked to the cholera toxin prophage.

We identify and characterize a gene cluster in El Tor Vibrio cholerae that encodes a cytotoxic activity for HEp-2 cells in vitro. This gene cluster contains four genes and is physically linked to the cholera toxin (CTX) element in the V. cholerae genome. We demonstrate by using insertional mutagenesis that this gene cluster is required for the cytotoxic activity. The toxin, RtxA, resembles members of the RTX (repeats in toxin) toxin family in that it contains a GD-rich repeated motif. Like other RTX toxins, its activity depends on an activator, RtxC, and an associated ABC transporter system, RtxB and RtxD. In V. cholerae strains of the classical biotype, a deletion within the gene cluster removes rtxC and eliminates cytotoxic activity. Other strains, including those of the current cholera pandemic, contain a functional gene cluster and display cytotoxic activity. Thus, the RTX gene cluster in El Tor O1 and O139 strains might have contributed significantly to their emergence. Furthermore, the RTX toxin of V. cholerae may be associated with residual adverse properties displayed by certain live, attenuated cholera vaccines.

Actinobacillus actinomycetemcomitans leukotoxin induces apoptosis in HL-60 cells.

Actinobacillus actinomycetemcomitans leukotoxin (Ltx) is a member of the repeats-in-toxin (RTX) family of pore-forming toxins and kills human immune cells. Currently, it remains unclear whether toxin-mediated killing of target cells involves the induction of necrosis or apoptosis. Therefore, the goal of this investigation was to determine whether Ltx is capable of causing apoptotic cell death in toxin-sensitive promyelocytic HL-60 cells. Multiparameter flow cytometric analysis of toxin-treated cells stained with Hoechst 33258 (or 33342) and 7-aminoactinomycin D allowed us to identify four populations: viable cells, early apoptotic cells, late apoptotic and/or secondarily necrotic cells, and a final population that was composed of cellular debris. Compared with control cells, HL-60 cells treated with Ltx exhibited a gradual decrease in forward light scatter with a coincident increase in side light scatter, indicative of a decrease in cell size and organelle condensation, respectively. Additional experiments demonstrated that Ltx-treated cells showed evidence of internucleosomal DNA fragmentation and phosphatidylserine translocation. The results of our studies clearly demonstrate that Ltx can kill HL-60 cells by inducing apoptosis. We hypothesize that elimination of acute inflammatory cells via this mechanism plays a critical role in the pathogenesis of diseases caused by A. actinomycetemeomitans.

Actin enhances the haemolytic activity of Escherichia coli.

Actin is a major cytoskeletal protein of mammalian muscle and non-muscle cells. Exposure of cells to soluble factors that damage cell membranes results in the release of actin into the extracellular spaces. The alpha-haemolysin (HlyA) of Escherichia coli is the prototype RTX (repeat in toxin) toxin and is thought to be important in virulence because of its ability to lyse cells by formation of pores in the cell membrane. These studies were conducted to determine if actin influences growth and haemolytic activity of E. coli. Growth of E. coli in the presence of actin resulted in culture supernatant haemolytic activity that was 2.4-, 2.7- and 3.3-fold greater than that of E. coli grown in medium containing BSA, non-supplemented medium, or medium containing heat-denatured actin, respectively. The enhanced haemolytic activity occurred only when actin was present during the growth phase and there was no effect when actin was added to culture supernatants containing haemolysin. The increased haemolytic activity by actin was concentration-dependent, detectable in early-exponential-phase growth, and associated with increased concentrations of secreted HlyA by Western blotting. Actin induced a 2.9-fold increase in alkaline phosphatase activity in E. coli CC118 with a TnphoA insertion in the hlyB determinant of the recombinant haemolysin plasmid pWAM04. These results indicate that extracellular actin enhances haemolysin production by E. coli and may have implications in the pathogenesis of E. coli infections.

The regulatory effect of fermentable sugar levels on the production of leukotoxin by Actinobacillus actinomycetemcomitans

The relationship between sugar availability and RTX (repeats in toxin) cytotoxin (leukotoxin) production in the periodontopathic bacterium, Actinobacillus actinomycetemcomitans, was investigated using a chemostat. A. actinomycetemcomitans 301-b produced significant amounts of leukotoxin in anaerobic fructose-limited chemostat cultures at a dilution rate of 0.15 h −1 and at pH 7.0. When the growth limitation was relieved by pulsing the cultures with 50 or 150 mM fructose (final concentrations), leukotoxin production immediately stopped and the amount of cellular leukotoxin decreased until the culture was returned to fructose-limited conditions. Leukotoxin synthesis was also repressed in the chemostat cultures by pulsing with glucose but not with the non-fermentable sugar analog, α-methyl- d-glucoside. Leukotoxin production was also repressed by fructose in chemostat cultures of ATCC 33384, which is generally recognized as a non-leukotoxin-producing or minimally leukotoxic strain.

Pore-forming properties of the plasmid-encoded hemolysin of enterohemorrhagic Escherichia coli O157:H7.

Lipid bilayer experiments were performed with the plasmid-encoded hemolysin of enterohemorrhagic Escherichia coli (EHEC) O157:H7 strain EDL933. EHEC-hemolysin caused the formation of transient ion-permeable channels by integration in lipid bilayer membranes composed of asolectin, dioleoylglycerophosphoethanolamine and phosphoserine but not of diphytanoylglycerophosphocholine. Channel formation showed the same characteristics when culture supernatants of E. coli strains EDL 933 or HB101/pEO40, precipitated or purified EHEC-hemolysin were used for these experiments. The EHEC-hemolysin channels had two different states at small transmembrane potential (20 mV): a prestate that represented the first step of channel formation (single-channel conductance 40 pS in 0.15 M KCl) and an open state (550 pS in 0.15 M KCl at pH 6.0). Experiments with different salts suggested that the EHEC-hemolysin-induced channels were cation-selective at neutral pH. The mobility sequence of the cations within the channels resembles their mobility sequence in the aqueous phase. The single-channel data were consistent with the formation of wide, water-filled channels by the EHEC hemolysin. The single channel conductance was strongly pH dependent and increased over 2.5-fold in the pH range 5-8. The analysis of the single-channel data using the Renkin correction factor suggested that the EHEC-hemolysin formed channels with an average diameter of 2.6 nm. This size could be confirmed by the results of osmotic-protection experiments. Neither sucrose nor raffinose inhibited toxin-dependent hemolysis, whereas hemolysis did not occur in the presence of dextran 4 (molecular mass, 4 kDa). Our results demonstrate that EHEC-hemolysin can be considered to be a highly active repeats-in-toxin (RTX)-toxin with a similar but not identical pore-forming capacity as the chromosomal encoded E. coli alpha-hemolysin.

Analysis of the EHEC hly operon and its location in the physical map of the large plasmid of enterohaemorrhagic Escherichia coli O157:h7.

Almost all clinical enterohaemorrhagic Escherichia coli (EHEC) O157:H7 isolates harbour a large virulence plasmid designated pO157. In this study, pO157 of EHEC O157:H7 reference strain EDL 933 was characterized at the molecular level. A restriction map was constructed by using seven restriction enzymes, with appropriate gel electrophoretic and hybridization methods. The molecular size of pO157 was determined to be 93.6 kb. By sequencing the DNA region extending in the 3'-direction of the previously described EHEC hlyC and hlyA genes, two further genes were discovered and analysed; these were termed EHEC hlyB and EHEC hlyD. The newly discovered genes together with the EHEC hlyC and hlyA genes constitute a typical RTX (Repeats in ToXin) determinant (EHEC hly operon) with the gene order CABD. The map position of the operon was determined by hybridization experiments. Analysis of a DNA fragment carrying the downstream flanking region of the EHEC hly operon revealed an open reading frame which was highly homologous to orf1 of RepFIB, a basic replicon of IncF plasmids. It was located close to the EHEC hly operon.

Interaction of Calcium with Bordetella pertussis Adenylate Cyclase Toxin: CHARACTERIZATION OF MULTIPLE CALCIUM-BINDING SITES AND CALCIUM-INDUCED CONFORMATIONAL CHANGES

The adenylate cyclase (CyaA) secreted by Bordetella pertussis is a toxin that is able to enter eukaryotic cells and cause a dramatic increase in cAMP level. In addition, the toxin also exhibits an intrinsic hemolytic activity that is independent from the ATP cycling catalytic activity of the toxin. Both the cytotoxic and hemolytic activities are calcium-dependent. In this work, we have analyzed the calcium interacting properties of CyaA. We have shown that CyaA exposed to CaCl2 could retain membrane binding capability and hemolytic activity when it was further assayed in the presence of an excess of EGTA. Determination of the calcium content of CyaA exposed first to calcium and subsequently to EGTA indicated that some (3-5) calcium ions remained bound to the protein, suggesting the existence of Ca2+ binding sites of high affinity. Binding of Ca2+ to these sites might be necessary for both the membrane binding capability and the hemolytic activity of the toxin. In addition, CyaA possesses a large number (about 45) of low affinity (KD = 0.5-0.8 mM) Ca2+ binding sites that are located in the C terminus of the toxin, between amino acids 1007 and 1706. This region mainly consists of about 45 repeated sequences of the type GGXGXDXLX (where X represents any amino acid) that are characteristic of the RTX (Repeat in ToXin) bacterial protein family. Our data suggest that each one can bind one calcium ion. Circular dichroism spectroscopy analysis showed that calcium binding to the low affinity sites induces a large conformational change of CyaA, as revealed by an important increase in the content of alpha-helical structures. This conformational change might be directly involved in the Ca(2+)-dependent translocation of the catalytic domain of CyaA through the plasma membrane of target cells.

Identification by in vitro complementation of regions required for cell-invasive activity of Bordetella pertussis adenylate cyclase toxin.

The adenylate cyclase toxin (CyaA) of Bordetella pertussis is a 1706-residue protein composed of an amino-terminal adenylate cyclase (AC) domain linked to a 1300-residue channel-forming RTX (repeats in toxin) haemolysin. The toxin delivers its AC domain into a variety of eukaryotic cells and impairs cellular functions by catalysing unregulated synthesis of cAMP from intracellular ATP. We have examined toxin activities of a set of deletion derivatives of CyaA. The results indicate that CyaA does not have a dedicated target cell-binding domain and that structural integrity and co-operation of all domains, as well as the post-translational fatty acylation mediated by an accessory protein CyaC, are all essential for target cell association and toxin activity of CyaA. When tested individually, all toxin derivatives were inactive and impaired in the tight association with the target cell surface. However, pairs of constructs with nonoverlapping deletions complemented each other in vitro and exhibited a partially restored cytotoxic activity. This suggests that at least a part of the active toxin may act in the form of dimers or higher oligomers. The complementation analysis revealed that the last 217 residues of CyaA, containing the unprocessed secretion signal, form an autonomous domain essential for toxin activity, and that the region from residue 624 to 780 may be directly involved in delivery of the AC toxin into cells.

Actinobacillus actinomycetemcomitans leukotoxin forms large conductance, voltage-gate ion channels when incorporated into planar lipid bilayers

Actinobacillus actinomycetemcomitans leukotoxin is a member of the bacterial RTX (repeats in toxin) toxin family, produced by a diverse group of Gram-negative pathogens. Members of this group of toxins, although similar in sequence, differ in target cell specificity with Actinobacillus actinomycetemcomitans leukotoxin demonstrating a unique species-and cell-type specificity. Purified A. actinomycetemcomitans leukotoxin added to pre-formed POPE/POPS lipid bilayers showed no spontaneous incorporation (to concentrations of 250 ng/ml). Reproducible channel activity was seen when the bilayer was reformed from lipid monolayers in the presence of toxin (50 ng/ml) in one of the aqueous chambers. Control experiments with heat-inactivated toxin did not display channel activity under the same experimental conditions. The channel behavior showed a complex pattern of multiple conductance levels of 118 262 and 406 pS in solutions containing 0.140 M NaCL The first two states showed voltage-dependent channel gating with approximately equal but opposite apparent gating charges of 1.4 electrons. A model accounting for the multiple conducting states and gating properties is presented.

Identification and immunological characterization of the domain of Actinobacillus actinomycetemcomitans leukotoxin that determines its specificity for human target cells.

Although extensive amino acid homology exists among the various Repeats in ToXin (RTX) family of bacterial cytolysins, the cellular and species specificities remain unique for individual toxins (i.e. Actinobacillus actinomycetemcomitans leukotoxin (LtxA) kills human monomyelocytes while a related toxin, Pasteurella hemeolytica leukotoxin (LktA) kills bovine lymphoid cells). To determine the Ltx domain responsible for species specificity, ltxA/lktA chimeric toxin genes were expressed in tandem with the ltxC gene under control of the P lambda promoter. The ability of lysates to kill either HL-60 (human) or BL-3 (bovine) cells was assessed by trypan blue exclusion. The critical area required for the chimeric toxins to recognize human target cells is a 253-amino acid fragment (residue 688-941) that contains the GGXGXDX(L[I[V[W[Y[F)X repeats. A panel of 12 neutralizing anti-LtxA monoclonal antibodies also recognized specificities within the 253-amino acid fragment. Epitope mapping of the monoclonal antibody panel showed that all antibodies bound to one of three sites on the LtxA molecule. One monoclonal recognized epitope A which was composed of LtxA residues 698-709 (KLDYYYTNKGFK), six antibodies recognized epitope B, a peptide composed of residues 746-757 (LIYGYDGDDRLY), whereas the remaining five monoclonals recognized epitope C, which is composed of residues 926-937 (DRARLKRQFELQ).

Structural and functional relationships among the RTX toxin determinants of gram-negative bacteria.

The RTX (repeats in toxin) cytolytic toxins represent a family of important virulence factors that have disseminated widely among Gram-negative bacteria. They are characterised by a series of glycine-rich repeat units at the C-terminal end of each protein. They also have other features in common. Secretion from the cell occurs without a periplasmic intermediate by a novel mechanism which involves recognition of a signal sequence at the C-terminus of the toxin by membrane-associated proteins that export the toxin directly to the outside of the cell. The structural gene for each protein encodes an inactive toxin which is modified post-translationally to an active cytotoxic form by another gene product before secretion. The genes for toxin synthesis, activation and secretion are for the most part grouped together on the chromosome and form an operon. The toxins all create pores in the cell membrane of target cells leading to eventual cell lysis and they appear to require Ca2+ for cytotoxic activity. Although the toxins have a similar mode of action, they vary in target cell specificity. Some are cytotoxic for a wide variety of eukaryotic cell types while others exhibit precise target cell specificity and are only active against leukocytes from certain host species. The characteristic glycine-rich repeat units have been identified in other exoproteins besides those with cytotoxic activity and it is likely that the novel secretory mechanism has been harnessed by a variety of pathogens to release important virulence-associated factors from the cell or to locate them on the cell surface.

Structural and functional relationships among the RTX toxin determinants of Gram-negative bacteria

The RTX (repeats in toxin) cytolytic toxins r represent a family of important virulence factors that have disseminated widely among Gram-negative bacteria. They are characterised by a series of glycine-rich repeat units at the C-terminal end of each protein. They also have other features in common. Secretion from the cell occurs without a periplasmic intermediate by a novel mechanism which involves recognition of a signal sequence at the C-terminus of the toxin by membrane-associated proteins that export the toxin directly to the outside of the cell. The structural gene for each protein encodes an inactive toxin which is modified post-translationally to an active cytotoxic form by another gene product before secretion. The genes for toxin synthesis, activation and secretion are for the most part grouped together on the chromosome and form an operon. The toxins all create pores in the cell membrane of target cells leading to eventual cell lysis and they appear to require Ca 2+ for cytotoxic activity. Although the toxins have a similar mode of action, they vary in target cell specificity. Some are cytotoxic for a wide variety of eukaryotic cell types while others exhibit precise target cell specificity and are only active against leukocytes from certain host species. The characteristic glycine-rich repeat units have been identified in other exoproteins besides those with cytotoxic activity and it is likely that the novel secretory mechanism has been harnessed by a variety of pathogens to release important virulence-associated factors from the cell or to locate them on the cell surface.