Translocation mediated by domain II of Pseudomonas exotoxin A: transport of barnase into the cytosol.

Pseudomonas exotoxin A (PE) is a protein toxin composed of three structural domains. Functional analysis of PE has revealed that domain I is the cell-binding domain and that domain III functions in ADP ribosylation. Domain II was originally designated as the translocation domain, mediating the transfer of domain III to the cytosol, because mutations in this domain result in toxin molecules with normal cell-binding and ADP-ribosylation activities but which are not cytotoxic. However, the results do not rule out the possibility that regions of PE outside of domain II also participate in the translocation process. To investigate this problem, we have now constructed a toxin in which domain III of PE is replaced with barnase, the extracellular ribonuclease of Bacillus amyloliquefaciens. This chimeric toxin, termed PE1-412-Bar, is cytotoxic to a murine fibroblast cell line and to a murine hybridoma resistant to the ADP-ribosylation activity of PE. A mutant form of PE1-412-Bar with an inactivating mutation in domain II at position 276 was significantly less toxic. Because the cytotoxic effect of PE1-412-Bar was due to the ribonuclease-activity of barnase molecules which had been translocated to the cytosol, we conclude that domain II of PE is not only essential but also probably sufficient to carry out the translocation process.     

A target-specific chimeric toxin composed of epidermal growth factor and Pseudomonas exotoxin A with a deletion in its toxin-binding domain

 We have fused the epidermal growth factor (EGF) to the amino terminus of Pseudomonas exotoxin A (PE) to create a cytotoxic agent, designated EGF-PE, which preferentially kills EGF-receptor-bearing cells. In this study, we analyzed the effect of the Ia domain, the binding domain, of PE on the cytotoxicity of EGF-PE towards EGF-receptor-bearing cells and tried to develop a more potent EGF-receptor-targeting toxin. EGF-PE molecules with sequential deletions at the amino terminus of PE were constructed and expressed in E. coli strain BL21(DE3). The cytotoxicity of these chimeric toxins was then examined. Our results show that the amino-terminal and carboxy-terminal regions of the Ia domain of PE are important for the cytotoxicity of a PE-based targeting toxin. To design a more potent PE-based EGF-receptor-targeting toxin, a chimeric toxin, named EGF-PE(Δ34–220), which had most of the Ia domain deleted but retained amino acid residues 1–33 and 221–252 of this domain, was constructed. EGF-PE(Δ34–220) has EGF-receptor-binding activity but does not show PE-receptor-binding activity and is mildly cytotoxic to EGF-receptor-deficient NR6 cells. As expected, EGF-PE(Δ34–220) is a more potent cytotoxic agent towards EGF-receptor-bearing cells than EGF-PE(Δ1–252), where the entire Ia domain of PE was deleted. In addition, EGF-PE(Δ34–220) was shown to be extremely cytotoxic to EGF-receptor-bearing cancer cells, such as A431, CE81T/VGH, and KB-3-1 cells. We also found that EGF-PE(Δ34–220) was highly expressed in BL21(DE3) and could be easily purified by urea extraction. Thus, EGF-PE(Δ34–220) can be a useful cytotoxic agent towards EGF-receptor-bearing cells. Received : 20 May 1994 / Received last revision : 9 September 1994 / Accepted : 28 September 1994     

A proper amino terminus of diphtheria toxin is important for cytotoxicity

A series of deletions and substitutions were made at the 5' end of the gene fusion between the first 388 codons of diphtheria toxin (DT) and a cDNA encoding human IL2. The chimeric protein (DT388-IL2) was expressed and purified from E. coli and found to be very cytotoxic to a human T cell line, HUT 102, that expresses a large number of IL2 receptors. Deletion of the first five amino acids of DT resulted in a non-cytotoxic chimeric protein that had both ADP-ribosylation activity and IL2 receptor binding activity. Deletion of the first two amino acids of DT had little effect on cytotoxicity, while deletion of the first four amino acids or of two acidic residues at positions 3 and 4 greatly reduced cytotoxicity. Unexpectedly, a mutant containing a single leucine in place of the first two amino acids (gly, ala) was 2-3 fold more active. The amino terminus of DT may participate in the translocation of the A chain to the cytosol in a manner similar to Pseudomonas exotoxin (PE) in which a specific C-terminal sequence has been proposed to be involved in its cytotoxicity.     

A chimeric toxin to study the role of the 21 kDa GTP binding protein rho in the control of actin microfilament assembly.

We have developed a new tool for studying the role of rho in actin stress fibre formation. Clostridium botulinum exoenzyme C3 which affects actin microfilament assembly by ADP-ribosylation of p21 rho was genetically fused in various ways to diphtheria toxin (DT). The resulting chimeric toxins were tested on Vero cells. Chimeras of C3 and both the A and B fragments of diphtheria toxin had reduced cell binding activities but were apparently able to penetrate into Vero cells by the same mechanism as DT. Upon exposure to low pH, DC3B, a fusion protein of C3 and DT B fragment, had a high affinity for the DT receptor, but was apparently not able to translocate to the cytosol upon acidification. In spite of this, addition of picomolar concentrations of DC3B to the growth medium caused disruption of the cell microfilament system associated with vinculin and blocked cell growth efficiently, indicating that the C3 part of DC3B reached the cytosol, albeit by a different mechanism than that of whole diphtheria toxin. The chimeric DC3B toxin was also applied to Vero cells infected by Listeria monocytogenes, a pathogenic bacterium that uses an unknown mechanism of actin polymerization to move rapidly in the cytosol. DC3B inhibited the bacterially induced microfilament assembly indicating that L. monocytogenes utilizes a cellular rho dependent mechanism in this process.     

Alanine scanning mutagenesis identifies surface amino acids on domain II of Pseudomonas exotoxin required for cytotoxicity, proper folding, and secretion into periplasm.

Pseudomonas exotoxin A (PE) is a single polypeptide chain that contains 613 amino acids and is arranged into three major structural domains. Domain Ia is responsible for cell recognition, domain II for translocation of PE across the membrane, and domain III for ADP-ribosylation of elongation factor 2. Recombinant PE can be produced in Escherichia coli and is efficiently secreted into the periplasm when an OmpA signal sequence is present. To investigate the role of the amino acids located on the surface of domain II in the action of the toxin against mammalian cells, we substituted alanine for each of the 27 surface amino acids present in domain II. Surprisingly, all 27 mutant proteins had some alteration in cytotoxicity when tested on human A431 or MCF7 cells or mouse L929 cells. Native PE has a compact structure and therefore is relatively protease resistant and very little ADP-ribosylation activity is detected in the absence of the denaturing agents like urea and dithiothreitol. Several of the mutations resulted in altered protease sensitivity of the toxin. Seven of the mutant molecules exhibited ADP-ribosylation activity without urea and dithiothreitol, indicating they are partially unfolded. Out of these seven mutants, six had increased cytotoxic activity on at least one of the target cell lines and the other retained its native cytotoxic potency.     

Comparative studies of the biological activities of human tumor necrosis factor and its derivatives

Biological activities of human tumor necrosis factor (TNF) and its derivatives were compared. In cytotoxicity assay with L929 cells, one derivative, designated as TNF(Asn), showed significantly lower activity than any other TNF examined. In binding assay, this derivative was also shown to have lower affinity for TNF receptors on L929 cells, suggesting that the cytotoxic activity of TNFs on L929 cells correlates with their affinity for receptors. We also found that the cytotoxic activity of TNF on A673 cells and its inhibitory effect on lipoprotein lipase were parallel with the cytotoxic activity on L929 cells, but the growth-enhancing activity on FS-4 cells and the cytotoxic activity on endothelial cells were not. It was also shown that TNF(Asn) had lower affinity than any other TNF for receptors on these target cells tested. These results suggested that there might be at least two types of cellular responses to TNF; one might correlate with the receptor-binding affinity of TNFs and the other not.     

Rational design of a chimeric toxin: an intramolecular location for the insertion of transforming growth factor alpha within Pseudomonas exotoxin as a targeting ligand.

To investigate the potential utility of Pseudomonas exotoxin (PE) in forming rationally designed chemotherapeutic agents, we inserted a cDNA encoding transforming growth factor alpha (TGF alpha) at several locations in a gene encoding a mutant full-length PE (PE4E) which does not bind to the PE receptor. After expression in Escherichia coli, we purified the chimeric toxins to near homogeneity and showed that they were specifically cytotoxic to human epidermoid, ovarian, colon, and hepatocellular carcinoma lines. Like the previously reported TGF alpha-PE40, one of the new molecules (TGF alpha-PE4E) contains the ligand at the amino terminus. Two additional chimeras (PE4E-TGF alpha and PE4E-TGF alpha-598-613) each contain TGF alpha inserted near the carboxyl terminus of PE. We show that preservation of the correct PE carboxyl-terminal amino acid sequence, REDLK, allows the toxins containing TGF alpha carboxyl inserts to retain significant cytotoxicity against target cells, since another molecule (PE4E-TGF alpha-ILK) containing a nonfunctional carboxyl-terminal sequence was over 100-fold less active. The chimeric toxins with TGF alpha had the same binding affinity for the EGF receptor whether the ligand occupied the amino or carboxyl position. Molecules with TGF alpha near the carboxyl position were consistently less active against target cells but also less toxic to mice than those with TGF alpha at the amino terminus, indicating both types of molecules might be therapeutically effective. Our results establish that a ligand can be placed near the carboxyl terminus of PE, within the portion of the toxin that translocates to the cytosol. The amino-terminal position in such molecules is then available for the placement of other targeting ligands.     

A nuclear localization sequence endows human pancreatic ribonuclease with cytotoxic activity

Some members of the ribonuclease superfamily, such as Onconase, are cytotoxic to cancer cells. This is not the case for human pancreatic ribonuclease. This lack of cytotoxicity is probably a result of the inhibition exerted by the cytosolic ribonuclease inhibitor once the protein has reached the cytosol. Until now, all cytotoxic human pancreatic ribonuclease variants have been described as being resistant to the inhibitor. Here, we report on the characterization of a cytotoxic variant of human pancreatic ribonuclease which has an Arg triplet introduced onto one of its surface-exposed loops. Despite its sensitivity to the inhibitor, this variant, called PE5, was only 5-15 times less cytotoxic than Onconase. When it was taken up by cells, it was only observed within late compartments of the endocytic pathway, probably because the number of molecules transported to the cytosol was too small to allow their visualization. Nuclear import assays showed that the Arg triplet endows PE5 with a nuclear localization signal. In these experiments, PE5 was efficiently transported to the nucleus where it was initially localized in the nucleolus. Although the Arg introduction modified the net charge of the protein and somehow impaired recognition by the cytosolic inhibitor, control variants, which had the same number of charges or were not recognized by the inhibitor, were not toxic. We concluded that targeting a ribonuclease to the nucleus results in cytotoxicity. This effect is probably due to ribonuclease interference with rRNA processing and ribosome assembly within the nucleolus.     

Growth inhibition and cytotoxicity of tumor necrosis factor in L929 cells is enhanced by high cell density and inhibition of mRNA synthesis

L929 cells were growth-inhibited after 1 to 2 days of treatment with human recombinant tumor necrosis factor (rTNF). This effect of rTNF was largely reversible, and L929 cells resumed normal growth when rTNF was removed. The rTNF showed growth inhibitory and cytotoxic activity when L929 cells approached a high cell density and grew slowly. This was shown in experiments in which L929 cells approached confluency at different times after being seeded at increasing initial densities. The rTNF had little effect on the growth of cells seeded at the lowest density tested. L929 cells cultured to high density synthesized RNA at a reduced rate. This suggested that a reduced rate of RNA synthesis may be at least in part responsible for the growth inhibitory and cytotoxic activities of rTNF on cells grown to high density. Treatment with inhibitors of RNA synthesis potentiated the cytotoxic activity of rTNF. Inhibition of mRNA synthesis was apparently responsible for the enhanced sensitivity to rTNF, as shown by experiments with 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole, an inhibitor of the synthesis of poly(A)-containing RNA.     

Substitution of foreign protein sequences into a chimeric toxin composed of transforming growth factor alpha and Pseudomonas exotoxin.

TGF alpha-PE40 is a chimeric toxin made by replacing domain Ia of Pseudomonas exotoxin (PE) with transforming growth factor alpha (TGF alpha). We have now replaced a portion of domain Ib of PE with different polypeptides or an extra domain III of PE in transforming growth factor alpha-PE40 and maintained cell killing. Thus, TGF alpha-PE40 can be used to transport foreign protein sequences into the cytosol of cells.     

Entry of diphtheria toxin linked to concanavalin A into primate and murine cells

Diphtheria toxin linked by a disulfide bridge to concanavalin A was highly toxic to HeLa S3 and Vero cells, as well as to murine L cells. The cells could be protected with alpha-methyl mannoside, indicating that the conjugate binds mainly through its concanavalin A moiety. Treatment of Vero cells with phospholipase C, TPA (12-O-tetradecanoylphorbol-13-acetate), and vanadate, which strongly reduce the ability of the cells to bind free diphtheria toxin, had little protective effect against the conjugate, whereas SITS (L-acetamido-4'-isothiocyano-stilbene-2,2'disulfonic acid), which inhibits diphtheria toxin binding, as well as the subsequent entry, protected Vero cells, but not L cells. Both types of cells are protected against the conjugate by NH4Cl and monensin, indicating that an acidified compartment is necessary for entry into the cytosol. Exposure of cells, bound with surface conjugate, to low pH induced entry of the toxin into Vero cells, but not into L Cells. Phospholipase C, TPA, and vanadate did not protect L cells against the conjugate. It is concluded that toxin in the conjugate enters L cells by a route which involves low pH, but which is not identical to that in Vero cells.     

Analysis of sequences in domain II of Pseudomonas exotoxin A which mediate translocation

Pseudomonas exotoxin (PE) contains 613 amino acids that are arranged into 3 structural domains. PE exerts its cell-killing effects in a series of steps initiated by binding to the cell surface and internalization into endocytic vesicles. The toxin is then cleaved within domain II near arginine-279, generating a C-terminal 37-kDa fragment that is translocated into the cytosol where it ADP-ribosylates elongation factor 2 and arrests protein synthesis. In this study, we have focused on the functions of PE which are encoded by domain II. We have used the chimeric toxin TGF alpha-PE40 to deliver the toxin's ADP-ribosylating activity to the cell cytosol. Deletion analysis revealed that sequences from 253 to 345 were essential for toxicity but sequences from 346 to 364 were dispensable. Additional point mutants were constructed which identified amino acids 339 and 343 as important residues while amino acids 344 and 345 could be altered without loss of cytotoxic activity. Our data support the idea that domain II functions by first allowing PE to be processed to a 37-kDa fragment and then key sequences such as those identified in this study mediate the translocation of ADP-ribosylation activity to the cytosol.     

Endosomes and toxin translocation

In this review we discuss data obtained by our group regarding the entry of toxins, especially ricin, diphtheria toxin (DT) and Pseudomonas exotoxin A (PE) into animal cells. We studied the translocation process of these toxins using endosomes purified from lymphocytes. This process is rate-limiting for toxicity and enables these toxins to reach the cytosol where they will inactivate the protein synthesis system and kill the cell. We could show that each of these toxins uses a different strategy to cross the endosome membrane. Whereas ricin transmembrane transport only relies on cytosolic ATP hydrolysis, PE first requires exposure to the low endosomal pH (pH-6), presumably to insert into the endosome membrane, before being translocated via a process which also requires cytosolic ATP hydrolysis. DT translocation is directly triggered and energized by the endosome-cytosol pH gradient. Using conjugates with dihydrofolate reductase we could indirectly show that ricin and PE require unfolding for translocation. A deletion approach enabled to produce a more cytotoxic PE mutant by increasing its translocation activity.     

Stress protection by a fluorescent Hsp27 chimera that is independent of nuclear translocation or multimeric dissociation

A chimeric protein consisting of enhanced green fluorescent protein (EGFP) fused to the N-terminus of human Hsp27 conferred stress protection in human A549 lung carcinoma and murine L929 cells that were stably transfected to express the chimera constitutively. The resultant protection was comparable with that in the same cell lines when they were transfected to express corresponding levels of Hsp27. Unlike L929 cells, A549 cells exhibit endogenous Hsp27 expression, whose expression was inhibited in proportion to the amount of fluorescent chimera expressed, suggesting that the A549 cells recognized the latter as Hsp27. Upregulation of Hsp27 or chimeric Hsp27 in all transfected cell lines (stable or transient transfection) caused no measurable change in cellular glutathione levels, indicating that glutathione played no role in the stress protection associated with either protein. Chimeric Hsp27 had a monomeric molecular weight of 55 kDa (that of Hsp27 plus EGFP) in both cell types and formed a 16-mer complex twice as massive as that formed by Hsp27. Heat shock or sodium arsenite induced phosphorylation of both chimeric Hsp27 and Hsp27, which resulted in the disaggregation of Hsp27 multimers in both cell types and disaggregation of 20% of the chimeric multimers in L929 cells. But chimeric Hsp27 multimers did not disaggregate after stress in A549 cells. Epifluorescence and confocal microscopy demonstrated that chimeric Hsp27 was restricted to the cytoplasm under normal growth conditions and after heat shock in all cells. This study supports the conclusions that Hsp27 stress protection requires neither its translocation into the nucleus nor the dissociation of its multimeric complex. Furthermore, it demonstrates that fluorescent chimeras of heat shock proteins can be functional and used to observe the protein's distribution within living cells.     

Characterization of a transferrin-diphtheria toxin conjugate

We report here the synthesis and properties of a hybrid toxin prepared by covalently coupling diphtheria toxin to transferrin. The purified material contained two major hybrid protein species and was highly cytotoxic to mouse LMTK- cells in culture, reducing protein synthesis by 50% in 24 h at a concentration of 1 ng/ml. Cytotoxic activity was completely abolished in the presence of exogenous transferrin or anti-transferrin or anti-diphtheria toxin, thus demonstrating that the hybrid toxin was intoxicating cells via their transferrin receptors and that both the diphtheria toxin and transferrin components of the conjugate were necessary for activity. NH4Cl, a drug that elevates the pH within acidic intracellular vesicles, also blocked cytotoxic activity, suggesting that a low intravesicular pH was required for activity. The inhibitory effect of NH4Cl could be abolished by exposing toxin-treated cells to acidic culture medium, further implicating an acid-dependent step in the mechanism of the hybrid toxin action. Studies on the kinetics of intoxication also implied that endocytosis and exposure to a low pH within vesicles were necessary for cytotoxicity. Altogether, the results suggest that the transferrin-diphtheria toxin conjugate binds to transferrin receptors and is internalized into acidic endocytic vesicles. The enzymatic moiety of diphtheria toxin then apparently enters the cytosol in response to the low pH and subsequently arrests protein synthesis.     

A recombinant diphtheria toxin related human CD4 fusion protein specifically kills HIV infected cells which express gp120 but selects fusion toxin resistant cells which carry HIV.

A chimeric toxin made by a genetic fusion between the DNA encoding the 389 N-terminal amino acids of diphtheria toxin and that coding for the V1 and V2 domains of human CD4 (amino acids 1-178) was produced, purified and examined for ADP ribosylation activity, gp120 binding and effects on acutely and chronically HIV infected cells. The fusion toxin DAB389CD4 possesses enzymatic activity and binds to gp120. DAB389CD4 was found to kill CEM and U937 cells infected by HIV selectively and efficiently in a dose dependent manner, however, fusion toxin treatment did not eliminate the virus from acutely infected cell cultures. In addition, treatment of chronically infected cells with DAB389CD4 rapidly led to the appearance of HIV infected cells which were resistant to the chimeric toxin. The experimental results reported here suggest that the potential use of gp120 targeted cytotoxic agents for the treatment of HIV infection should be viewed with caution.     

Pseudomonas exotoxin exhibits increased sensitivity to furin when sequences at the cleavage site are mutated to resemble the arginine-rich loop of diphtheria toxin

To be toxic for mammalian cells, Pseudomonas exotoxin (PE) requires proteolytic cleavage between Arg-279 and Gly-280. Cleavage, which is mediated by the cellular protease furin, generates an active C-terminal fragment which translocates to the cytosol and inhibits protein synthesis. In vitro , furin-mediated cleavage is optimal at pH 5.5 with a relatively slow turnover rate. Within cells, only 5–10% of cell-associated PE is cleaved. To investigate the reasons for this inefficient cleavage, the amino acid composition near the cleavage site was altered to resemble more closely the arginine-rich sequence from the functionally similar region of diphtheria toxin (DT). Four PE-DT mutants were generated, whereby 1, 5, 6 or 8 amino acids at the PE-cleavage site were changed to amino acids found at the DT-cleavage site. Mutant proteins were expressed in Escherichia coli , purified and then analysed for their susceptibility to cleavage by furin and trypsin, susceptibility to cell-mediated cleavage, and cytotoxic activity relative to wild-type PE. At pH 5.5, the rate of both furin-mediated cleavage and trypsin-mediated cleavage increased dramatically when amino acids in PE were altered to resemble the DT sequence. This increase did not alter the pH optimum for furin-mediated cleavage of PE toxins, which remained at pH 5.0–5.5. When radioactive versions of selected PE-DT proteins were added to intact cells, an increase in the percentage of molecules that were cleaved relative to wild-type PE was also seen. However, changes that favoured increased proteolysis apparently interfered with other important toxin functions because none of the PE-DT proteins exhibited enhanced toxicity for cells when compared with the activity of wild-type PE.     

Requirement of specific receptors for efficient translocation of diphtheria toxin A fragment across the plasma membrane

The role of specific receptors in the translocation of diphtheria toxin A fragment to the cytosol and for the insertion of the B fragment into the cell membrane was studied. To induce nonspecific binding to cells, toxin was either added at low pH, or biotinylated toxin was added at neutral pH to cells that had been treated with avidin. In both cases large amounts of diphtheria toxin became associated with the cells, but there was no increase in the toxic effect. There was also no increase in the amount of A fragment that was translocated to the cytosol, as estimated from protection against externally added Pronase E. In cells where specific binding was abolished by treatment with 12-O-tetradecanoyl-phorbol 13-acetate, trypsin, or 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid, unspecific binding did not induce intoxication or protection against protease. This was also the case in untreated L cells, which showed no specific binding of the toxin. When Vero cells with diphtheria toxin bound to specific receptors were exposed to low pH, the cells were permeabilized to K+, whereas this was not the case when the toxin was bound nonspecifically at low pH or via avidin-biotin. The data indicate that the cell-surface receptor for diphtheria toxin facilitates both insertion of the B fragment into the cell membrane and translocation of the A fragment to the cytosol.     

Cytotoxic lymphokines produced by cloned human cytotoxic T lymphocytes. I. Cytotoxins produced by antigen-specific and natural killer-like CTL are dissimilar to classical lymphotoxins

Several cloned lines of IL 2-dependent human T cells derived from alloantigen, mitogen, or IL 2-stimulated peripheral blood lymphocytes were examined for their surface marker expression, cytolytic activity in a 51Cr-release assay, and capacity to release cytotoxic lymphokines. Thirty cell lines exhibiting either antigen-specific natural killer cell activity or lectin-dependent killer cell function, which expressed either the CD4 or CD8 surface differentiation markers, were capable of producing cytotoxin(s) in response to the lectins phytohemagglutinin and concanavalin A. Cytotoxin activity was detected on the murine L929 target cell in a 16-hr cytotoxicity assay. In contrast, several nonlytic T cell tumor lines failed to produce a soluble cytotoxin. Antibodies capable of neutralizing human alpha-lymphotoxin were completely ineffective in inhibiting the cytotoxin(s) produced by any of the cytotoxic T lymphocytes (CTL) cell lines. Comparative gel filtration and HPLC hydrophobic chromatography of alpha-lymphotoxin and CTL toxin produced by the CTL-830.B2 clone revealed significant differences in their elution profiles. The CTL-produced toxin and alpha-lymphotoxin exhibited similar kinetics of lysis of the L929 target cells, with 50% target cell lysis occurring at 10 hr. These data indicate human CTL produce a cytotoxin(s) antigenically distinct from alpha-lymphotoxin and imply that human cytolytic effector T cells are not the cellular source for the production of human alpha-lymphotoxin. The relationship of alpha-lymphotoxin and CTL toxin production was investigated in unseparated peripheral blood mononuclear cells stimulated with lectins or IL 2 for 1 and 5 days. Anti-alpha-lymphotoxin antibodies were capable of neutralizing only 30 to 50% of the cytotoxic activity in 24-hr supernatants. Cytotoxic activity in supernatants harvested after 120 hr stimulation with PHA or Con A was neutralized 70 to 100%, whereas the toxin(s) released from IL 2-stimulated lymphocytes was only neutralized 30%. These data suggest the observed heterogeneity of cytotoxic lymphokines produced by unseparated mononuclear cells depends in part on the subpopulations of effector cells responding to a given stimulus and the capacity of different subpopulations to produce distinct cytotoxins.     

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.