A periplasmic intermediate in the extracellular secretion pathway of Pseudomonas aeruginosa exotoxin A.

Pseudomonas aeruginosa exotoxin A is synthesized with a secretion signal peptide typical of proteins whose final destination is the periplasm. However, exotoxin A is released from the cell without a detectable periplasmic pool, suggesting that additional determinants in this protein are important for recognition by a specialized machinery of extracellular secretion. The role of the N terminus of the mature exotoxin A in this recognition was investigated. A series of exotoxin A proteins with amino acid substitutions for the glutamic acid pair at the +2 and +3 positions were constructed by mutagenesis of the exotoxin A gene. These N-terminal acidic residues of the mature exotoxin A protein were found to be important not only for efficient processing of the precursor protein but also for extracellular localization of the toxin. The mutated exotoxin A proteins, in which a glutamic acid at the +2 position was replaced by a lysine or a double substitution of lysine and glutamine for the pair of adjacent glutamic acids, accumulated in precursor forms in the mixed cytoplasmic and membrane fractions, which was not seen with the wild-type exotoxin A. The processing of the precursor form of one exotoxin A mutant, in which the glutamic acid at the +2 position was replaced with a glutamine, was not affected. Moreover, a substantial fraction of the mature forms of all three mutants of exotoxin A accumulated in the periplasm, while wild-type exotoxin A could be detected only extracellularly. The periplasmic pools of these variants of exotoxin A could therefore represent the intermediate state during extracellular secretion. The signal for extracellular localization may be located in a small region near the amino terminus of the mature protein or could consist of several regions that are brought together after the polypeptide has folded. Alternatively, the acidic residues may be important for ensuring a conformation essential for exotoxin A to traverse the outer membrane.     

A specific targeting domain in mature exotoxin A is required for its extracellular secretion from Pseudomonas aeruginosa.

A number of Gram-negative bacteria, including Pseudomonas aeruginosa, actively secrete a subset of periplasmic proteins into their surrounding medium. The presence of a putative extracellular targeting signal within one such protein, exotoxin A, was investigated. A series of exotoxin A truncates, fused to beta-lactamase, was constructed. Hybrid proteins, which carry at their N- termini 120, 255, 355 or the entire 613 residues of the mature exotoxin A, were stable and were secreted into the extracellular medium. Hybrid proteins which carry residues 1-30 and 1-60 of the mature exotoxin A were unstable; however, they could be detected entirely within the cells after a short labeling period. A hybrid with beta-lactamase was constructed which carried only the N-terminal residues 1-3 and region 60-120 of exotoxin A. It was also secreted into the culture medium, suggesting that a specific 60 amino acid domain contains the necessary targeting information for translocation of exotoxin A across the outer membrane. The secretion of the hybrid proteins is independent of the passenger protein, since a similar exotoxin A-murine interleukin 4 hybrid protein was also secreted. The extracellular targeting signal between amino acids 60 and 120 is rich in anti-parallel beta-sheets. It has been shown previously to be involved in the interaction of the exotoxin A with the receptors of the eukaryotic cells. In the three- dimensional view, the targeting region is on the toxin surface where it is easily accessible to the components of the extracellular secretion machinery.     

Protein export by a gram-negative bacterium: production of aerolysin by Aeromonas hydrophila.

The synthesis and export of aerolysin, an extracellular protein toxin released by the gram-negative bacterium Aeromonas hydrophila, was studied by pulse-labeling with [35S]methionine. The toxin was synthesized as a higher-molecular-weight precursor. This was processed cotranslationally, resulting in the appearance within the cell of the mature protein, which was then exported to the supernatant. Precursor aerolysin accumulated in cells incubated in the presence of carbonyl cyanide m-chlorophenyl hydrazone, a substance which also inhibited the export of mature aerolysin from the cell. The entrapped mature toxin could not be shocked from the cells, although it could be digested by protease applied to shocked cells. The toxin was processed and translocated across the inner membrane of pleiotropic export mutants and accumulated in the periplasm. The results indicate that more than one step is required for the export of the protein and that aerolysin does not cross the inner and outer membranes simultaneously.     

A dipeptide insertion in domain I of exotoxin A that impairs receptor binding

Deletions within the structural exotoxin A gene of 27 or 119 amino acids in domain I of the mature polypeptide, or of 88 or 105 amino acids in domains I and II, resulted in the synthesis of exotoxin A (ETA) polypeptides that were not secreted from Pseudomonas aeruginosa hosts but were localized in the cell membrane. Insertions of a hexanucleotide sequence, either pCGAGCT or pCGAATT, at TaqI sites within the gene resulted in variant exotoxin A polypeptides which were secreted normally. pCGAGCT causes insertion of either Glu-Leu or Ser-Ser in the amino acid sequence of the toxin, while pCGAATT causes insertion of either Glu-Phe or Asn-Ser dipeptides. Although the cytotoxicity of eight variants was unimpaired, that of four others was reduced, and one variant which had a Glu-Phe insert between residues 60 and 61 (ETA-60EF61) was 500-fold less cytotoxic than wild-type exotoxin A. Purified ETA-60EF61 dissociated much faster from mouse LMTK- cells than wild-type ETA, suggesting that the insertion impaired the ability of ETA-60EF61 to interact with exotoxin A receptors. The location of the insert is within a major concavity on the surface of domain I of the exotoxin A molecule, suggesting that this concavity is important for toxin-receptor interaction.     

Precursor in cotranslational secretion of diphtheria toxin.

By extracellular labeling of peptides of intact Corynebacterium diphtheriae, followed by fractionation of the cells and chain completion by isolated polysomes, it is shown that diphtheria toxin is formed and secreted cotranslationally by membrane-bound polysomes; free polysomes from none. Moreover, when the chains on these polysomes were completed in vitro, in the absence of membrane they were found to include not only diphtheria toxin of a molecular weight of 62,000, but also a larger precursor of a molecular weight of 68,000. The precursor was identified by several properties: immune precipitation; conversion into toxin fragments A and B; adenosine diphosphate ribosyl-transferase activity after activation with trypsin; and cleavage to 62,000 daltons by membrane enzymes. The precursor yields an N-terminal A fragment with a broadened molecular weight distribution, compared with that from authentic toxin, thus supporting the expectation that the extra segment of the precursor is N-terminal.     

Transport and processing of staphylococcal alpha-toxin

Two larger precursors to staphylococcal alpha-toxin were identified and partially characterized. Both precursor proteins were present on the cell membrane at very low levels and appeared to be rapidly processed to the mature form. Dinitrophenol inhibited processing such that the two precursors accumulated in the membranes, whereas little extracellular (mature) alpha-toxin is formed. The peptide maps of the 35S-labeled peptides from extracellular alpha-toxin and the two precursors were almost identical. The larger precursor protein contained four additional peptides and the smaller precursor protein contained three additional peptides not found in the extracellular toxin.     

Electrochemical potential releases a membrane-bound secretion intermediate of maltose-binding protein in Escherichia coli.

A secretionary intermediate of the Escherichia coli maltose-binding protein accumulated in the inner membrane when the membrane electrochemical potential was reduced and the cytosolic ATP concentration was normal. The intermediate was mature in size, but maintained a conformation similar to the cytosolic precursor form, and not the mature periplasmic protein, as measured by differences in susceptibility to proteinase K in vitro. The intermediate was located on the periplasmic side of the inner membrane. Restoration of the membrane electrochemical potential resulted in the movement of the intermediate from the inner membrane to the periplasm. In other experiments in which the ATP concentration was reduced by 96% and the electrochemical potential remained normal, no intermediate accumulated. Thus, the final step in the export of maltose-binding protein requires the electrochemical potential of the inner membrane and does not require ATP.     

Potent receptor-mediated cytotoxicity of granulocyte colony-stimulating factor-Pseudomonas exotoxin, a fusion protein against myeloid leukemia cells

A chimeric toxin in which the cell-surface binding domain of Pseudomonas exotoxin A was replaced with mature human granulocyte colony-stimulating factor (G-CSF) was produced in Escherichia coli, purified and tested for its biological activity on the human G-CSF-responsive myeloid leukemia cell line, UT7/GR. This fusion protein, termed G-CSF-PE40, showed potent cytotoxicity in the cell line in a dose-dependent manner. G-CSF-PE40 displaced binding of biotinylated G-CSF to its receptor, and the cytotoxicity of G-CSF-PE40 was neutralized by an excess of wild-type G-CSF, indicating the receptor-mediated effects of this chimeric toxin. When G-CSF-PE40 was injected into normal mice, they showed transient neutropenia but no significant changes in the numbers of red blood cells or platelets. Furthermore, G-CSF-PE40 prolonged the survival of mice transplanted with syngeneic myeloid leukemia cells. These observations suggest that G-CSF-PE40 may be useful in targeted therapy of myeloid leukemia cells expressing G-CSF receptors.     

Expression cloning of a diphtheria toxin receptor: identity with a heparin-binding EGF-like growth factor precursor.

A monkey cDNA (pDTS) encoding a diphtheria toxin (DT) sensitivity determinant was isolated by expression cloning in mouse L-M cells. Mouse cells are naturally resistant to DT, because they lack functional cell surface receptors for the toxin. Unlike wild-type L-M cells, pDTS-transfected mouse cells are extremely toxin sensitive and specifically bind radioiodinated DT. Intoxication of the transfected cells requires receptor-mediated endocytosis of the bound toxin. The cDNA is predicted to encode an integral membrane protein that is identical to the precursor of a heparin-binding EGF-like growth factor. The DT sensitivity protein is thus a growth factor precursor that DT exploits as a receptor.     

Modification of the cell surface with neuraminidase increases the sensitivities of cells to diphtheria toxin and Pseudomonas aeruginosa exotoxin.

When cell lines that are susceptible to diphtheria toxin, such as human FL cells, were treated with C. perfringens neuraminidase their sensitivities to the toxin were increased. The sensitivities of the cells to the toxin were also increased by treatment with neuraminidase from Arthrobacter ureafaciens or HVJ (Sendai virus). Neuraminidase did not have this effect on a toxin-resistant cell line. It also did not increase the cytotoxic effect of a large concentration of fragment A of diphtheria toxin, which lacks the moiety of the toxin molecule that binds to the cell membrane. Neuraminidase from C. perfringens or HVJ also increased the sensitivity of cells to ricin toxin. Furthermore, neuraminidase from C. perfringens or A. ureafaciens increased the sensitivity of cells to Pseudomonas aeruginosa exotoxin (PA toxin), but in this case neuraminidase from HVJ did not have a similar effect.     

Analysis of the structure-function relationship of Pseudomonas aeruginosa exotoxin A

Biochemical and genetic techniques have provided considerable insight into the structure-function relationship of one of the ADP-ribosyl transferases produced by Pseudomonas aeruginosa, exotoxin A. Exotoxin A contains a typical prokaryotic signal sequence which, in combination with the first 30 amino-terminal amino acids of the mature protein, is sufficient for exotoxin A secretion from P. aeruginosa. Determination of the nucleotide sequence and crystalline structure of this prokaryotic toxin allowed a molecular model to be constructed. The model reveals three structural domains of exotoxin A. Analysis of the identified domains shows that the amino-terminal domain (domain I) is involved in recognition of eukaryotic target cells. Furthermore, the central domain (domain II) is involved in secretion of exotoxin A into the periplasm of Escherichia coli. Evidence also implicates the role of domain II in translocation of exotoxin A from the eukaryotic vesicle which contains the toxin after it becomes internalized into susceptible eukaryotic cells via receptor-mediated endocytosis. The carboxy-terminal portion of exotoxin A (domain III) encodes the enzymatic activity of the molecule. The structure of this domain includes a cleft which is hypothesized to be the catalytic site of the enzyme. Several residues within domain III have been identified as having a direct role in catalysis, while others are hypothesized to play an important structural role.     

A class of mutant CHO cells resistant to cholera toxin rapidly degrades the catalytic polypeptide of cholera toxin and exhibits increased endoplasmic reticulum-associated degradation

After binding to the eukaryotic cell surface, cholera toxin undergoes retrograde transport to the endoplasmic reticulum. The catalytic A1 polypeptide of cholera toxin (CTA1) then crosses the endoplasmic reticulum membrane and enters the cytosol in a process that may involve the quality control mechanism known as endoplasmic reticulum-associated degradation. Other toxins such as Pseudomonas exotoxin A and ricin are also thought to exploit endoplasmic reticulum-associated degradation for entry into the cytosol. To test this model, we mutagenized Chinese hamster ovary cells and selected clones that survived a prolonged coincubation with Pseudomonas exotoxin A and ricin. These lethal endoplasmic reticulum-translocating toxins bind different surface receptors and target different cytosolic substrates, so resistance to both would likely result from disruption of a shared trafficking or translocation event. Here we characterize two Pseudomonas exotoxin A/ricin-resistant clones that exhibited increased endoplasmic reticulum-associated degradation. Both clones acquired the following unselected traits: (i) resistance to cholera toxin; (ii) increased degradation of an endoplasmic reticulum-localized CTA1 construct; (iii) increased degradation of an established endoplasmic reticulum-associated degradation substrate, the Z variant of alpha1-antitrypsin (alpha1AT-Z); and (iv) reduced secretion of both alpha1AT-Z and the transport-competent protein alpha1AT-M. Proteosome inhibition partially rescued the alpha1AT-M secretion deficiencies. However, the mutant clones did not exhibit increased proteosomal activity against cytosolic proteins, including a second CTA1 construct that was expressed in the cytosol rather than in the endoplasmic reticulum. These results suggested that accelerated endoplasmic reticulum-associated degradation in the mutant clones produced a cholera toxin/Pseudomonas exotoxin A/ricin-resistant phenotype by increasing the coupling efficiency between toxin translocation and toxin degradation.     

Nucleotide sequence of the streptococcal pyrogenic exotoxin type B gene and relationship between the toxin and the streptococcal proteinase precursor.

The streptococcal pyrogenic exotoxin (SPE) type B-encoding structural gene, speB, was subcloned from a 4.5-kilobase streptococcal DNA insert onto a 2.4-kilobase insert, which was then sequenced. Studies indicated that a 1,194-base-pair open reading frame encoded a 398-amino-acid protein. Removal of the putative signal peptide resulted in a mature protein with 371 residues (molecular weight, 40,314), which was subsequently proteolyzed to yield a 253-residue breakdown product (molecular weight, 27,588). This processing was confirmed by amino-terminal sequencing of both the 40,314-molecular-weight protein and the breakdown product. Monte Carlo analysis indicated that SPE B was relatively dissimilar to other members of the pyrogenic toxin family that also includes SPEs A and C, toxic shock syndrome toxin 1, and the staphylococcal enterotoxins. Comparison with the published amino acid sequence of streptococcal proteinase precursor as well as DNA hybridization experiments indicated that SPE B is a variant of this protein even though the particular gene sequenced did not encode a proteolytically active molecule.     

Membrane type-1 matrix metalloproteinase (MT1-MMP) protects malignant cells from tumoricidal activity of re-engineered anthrax lethal toxin

Protective antigen (PA) and lethal factor (LF) are the two components of anthrax lethal toxin. PA is responsible for interacting with cell receptors and for the subsequent translocation of LF inside the cell compartment. A re-engineered toxin comprised of PA and a fusion chimera LF/Pseudomonas exotoxin (FP59) is a promising choice for tumor cell surface targeting. We demonstrated, however, that in vitro in cell-free system and in cultured human colon carcinoma LoVo, fibrosarcoma HT1080 and glioma U251 cells membrane type-1 matrix metalloproteinase (MT1-MMP) cleaves both the PA83 precursor and the PA63 mature protein. Exhaustive MT1-MMP cleavage of PA83 in vitro generates several major degradation fragments with an N-terminus at Glu40, Leu48, and Gln512. In cultured cells, MT1-MMP-dependent cleavage releases the cell-bound PA83 and PA63 species from the cell surface. As a result, MT1-MMP expressing cells have less PA63 to internalize. In agreement, our observations demonstrate that MT1-MMP proteolysis of PA makes the MT1-MMP-expressing aggressive invasive cells resistant to the cytotoxic effect of a bipartite PA/FP59 toxin. We infer from our studies that synthetic inhibitors of MMPs are likely to increase the therapeutic anti-cancer effect of anthrax toxin. In addition, our study supports a unique role of furin in the activation of PA, thereby suggesting that furin inhibitors are the likely specific drugs for short-term therapy of anthrax infection.     

Cotranslational secretion of diphtheria toxin and alkaline phosphatase in vitro: involvement of membrane protein(s).

Crude messenger ribonucleic acid fractions isolated from Corynebacterium diphtheriae and Escherichia coli were translated in an E. coli in vitro protein-synthesizing system and yielded precursors of the secreted proteins diphtheria toxin and alkaline phosphatase, respectively. Addition of inverted E. coli inner membrane vesicles to the system during the initial stages of translation resulted in the intravesicular segregation of mature diphtheria toxin and alkaline phosphatase. Outer membrane vesicles or inner membrane vesicles whose cytoplasmic surfaces had been treated with pronase could not mediate transmembrane transfer of diphtheria toxin or alkaline phosphatase. However, inner membrane vesicles isolated from E. coli spheroplasts which had been treated with pronase and inner membrane vesicles complexed with ribosomes during pronase treatment were functional in transmembrane transfer. At temperatures below the phase transition of E. coli membranes, no intravesicular segregation of alkaline phosphatase or diphtheria toxin was observed. The precursor forms of each protein accumulated free from the vesicles. These results suggest that an inner membrane protein, exposed on the cytoplasmic surface, plays an integral role in secretion.     

p97 Is in a complex with cholera toxin and influences the transport of cholera toxin and related toxins to the cytoplasm

Certain protein toxins, including cholera toxin, ricin, and Pseudomonas aeruginosa exotoxin A, are transported to the lumen of the endoplasmic reticulum where they retro-translocate across the endoplasmic reticulum membrane to enter the cytoplasm. The mechanism of retrotranslocation is poorly understood but may involve the endoplasmic reticulum-associated degradation pathway. The AAA ATPase p97 (also called valosin-containing protein) participates in the retro-translocation of cellular endoplasmic reticulum-associated degradation substrates and is therefore a candidate to participate in the retrotranslocation of protein toxins. To investigate whether p97 functions in toxin delivery to the cytoplasm, we measured the sensitivity to toxins of cells expressing either wild-type p97 or a dominant ATPase-defective p97 mutant under control of a tetracycline-inducible promoter. The rate at which cholera toxin and related toxins entered the cytoplasm was reduced in cells expressing the ATPase-defective p97, suggesting that the toxins might interact with p97. To detect interaction, the cholera toxin A chain was immunoprecipitated from cholera toxin-treated Vero cells, and co-immunoprecipitation of p97 was assessed by immunoblotting. The immunoprecipitates contained both cholera toxin A chain and p97, evidence that the two proteins are in a complex. Altogether, these results provide functional and structural evidence that p97 participates in the transport of cholera toxin to the cytoplasm.     

Effects of Tetanus Toxin on Catecholamine Release from Intact and Digitonin-Permeabilized Chromaffin Cells

Abstract: Tetanus exotoxin inhibited Ca²⁺-dependent cate-cholamine secretion in a dose-dependent manner in digito-nin-permeabilized chromaffin cells. The inhibition was specific for tetanus exotoxin and the B fragment of tetanus toxin; the C fragment had no effect. Inhibition required the introduction of toxin into the cell, and was not seen when intact cells were preincubated with the toxin or toxin fragments. The degree of inhibition was related to the length of preincubation with toxin, as well as the concentration of toxin used. A short preincubation with toxin was sufficient to inhibit secretion, and the continued presence of toxin in the incubation medium was not required during the incubation with Ca²⁺. The inhibition of secretion by tetanus toxin or the B fragment was not overcome with increasing Ca²⁺ concentrations. Tetanus toxin also inhibited catechol-amine secretion enhanced by phorbol ester-induced activation of protein kinase C. Thus, the toxin or a proteolytic fragment of the toxin can enter digitonin-permeabilized cells to interact with a component of the Ca²⁺-dependent exocytotic pathway to inhibit secretion.     

Effect of reduced membrane lipid fluidity on the biosynthesis of lipopolysaccharide of Escherichia coli

A low molecular weight precursor of lipopolysaccharide was accumulated under conditions in which the membrane lipids of a fatty acid auxotroph of Escherichia coli were reduced to a non-fluid state. The lipopolysaccharide precursor was detected, by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and autoradiography, in membranes isolated from cells which were pulse-labeled with N-acetyl-[1-14C]glucosamine. The precursor could be chased into mature lipopolysaccharide by returning the membrane lipids to a normal fluid state. Conversion of the precursor to lipopolysaccharide was inhibited by the presence of potassium cyanide or sodium arsenate. The processing of several outer membrane protein precursors, including the promatrix proteins, was also inhibited under these conditions. Preliminary characterization of the lipopolysaccharide precursor was undertaken.     

Molybdenum requirement for translocation of dimethyl sulfoxide reductase to the periplasmic space in a photodenitrifier, Rhodobacter sphaeroides f. sp. denitrificans.

Translocation of dimethyl sulfoxide (DMSO) reductase to the periplasmic space was studied in vivo with a photodenitrifier, Rhodobacter sphaeroides f. sp. denitrificans, using immunoblotting analysis and radioactive labeling. A polypeptide with an apparent molecular mass about 2,000 Da higher than that of DMSO reductase accumulated during induction of the reductase with DMSO. An uncoupler, carbonyl cyanide-m-chlorophenylhydrazone, inhibited the processing of the polypeptide after cells had been radioactively pulse-labeled with [35S]methionine. These results indicated that the higher-molecular-mass polypeptide was the precursor form of DMSO reductase. The precursor form accumulated in either the cytoplasm or the membrane, whereas the mature form accumulated in the periplasmic space. The membrane-bound precursor was sensitive to proteinase K treatment from both the cytoplasmic and periplasmic sides of the membrane, indicating that the polypeptide binds to the membrane, exposing it to both the outer and inner surfaces of the cytoplasmic membrane. Processing of the precursor was hampered by removal of molybdate from the medium and was restored by its readdition. It was also inhibited by the addition of tungstate in the medium.