Identification of the region of rho involved in substrate recognition by Escherichia coli cytotoxic necrotizing factor 1 (CNF1).

The Escherichia coli cytotoxic necrotizing factor 1 (CNF1) and the Bordetella dermonecrotic toxin (DNT) activate Rho GTPases by deamidation of Gln(63) of RhoA (Gln(61) of Cdc42 and Rac). In addition, both toxins possess in vitro transglutaminase activity in the presence of primary amines. Here we characterized the region of Rho essential for substrate recognition by the toxins using Rho/Ras chimeras as protein substrates. The chimeric protein Ras55Rho was deamidated or transglutaminated by CNF1. Rat pheochromocytoma PC12 cells microinjected with Ras55Rho developed formation of neurite-like structures after treatment with the CNF1 holotoxin indicating activation of the Ha-Ras chimera and Ras-like effects in intact cells. The Ras59Rho78Ras chimera protein contained the minimal Rho sequence allowing deamidation or transglutamination by CNF1. A peptide covering mainly the switch II region and consisting of amino acid residues Asp(59) through Asp(78) of RhoA was substrate for CNF1. Changes of amino acid residues Arg(68) or Leu(72) of RhoA into the corresponding residues of Ras (R68ARhoA and L72QRhoA) inhibited deamidation and transglutamination of the mutants by CNF1. In contrast to CNF1, DNT did not modify Rho/Ras chimeras or the switch II peptide (Asp(59) through Asp(78)). Glucosylation of RhoA at Thr(37) blocked deamidation by DNT but not by CNF. The data indicate that CNF1 recognizes Rho GTPases exclusively in the switch II region, whereas the substrate recognition by DNT is characterized by additional structural requirements.     

Structure of the Rho-activating domain of Escherichia coli cytotoxic necrotizing factor 1.

Certain uropathogenic and neonatal meningitis-causing strains of Escherichia coli express a 114 kDa protein toxin called cytotoxic necrotizing factor 1 (CNF1). The toxin causes alteration of the host cell actin cytoskeleton and promotes bacterial invasion of blood-brain barrier endothelial cells. CNF1 belongs to a unique group of large cytotoxins that cause constitutive activation of Rho guanosine triphosphatases (GTPases), which are key regulators of the actin cytoskeleton. This group also includes E. coli cytotoxic necrotizing factor 2 (CNF2, 114 kDa) and dermonecrotic toxins (DNT, 159 kDa) of Bordetella spp. with related sequences occurring in Yersinia spp. Here we show that the catalytic region of CNF1 exhibits a novel protein fold as determined by its 1.83 A resolution crystal structure. The structure reveals that CNF1 has a Cys-His-main chain oxygen catalytic triad reminiscent of enzymes belonging to the catalytic triad superfamily. The position of the catalytic Cys residue at the base of a deep pocket restricts access to potential substrates and helps explain the high specificity of this and related toxins.     

A single amino acid substitution in the enzymatic domain of cytotoxic necrotizing factor type 1 of Escherichia coli alters the tissue culture phenotype to that of the dermonecrotic toxin of Bordetella spp

Cytotoxic necrotizing factor type 1 (CNF1) and dermonecrotic toxin (DNT) share homology within their catalytic domains and possess deamidase and transglutaminase activities. Although each toxin has a preferred enzymatic activity (i.e. deamidation for CNF1 and transglutamination for DNT) as well as target substrates, both modify a specific glutamine residue in RhoA, Rac1 and Cdc42, which renders these GTPases constitutively active. Here we show that despite their similar mechanisms of action CNF1 and DNT induced unique phenotypes on HEp-2 and Swiss 3T3 cells. CNF1 induced multinucleation of HEp-2 cells and was cytotoxic for Swiss 3T3 cells (with binucleation of the few surviving cells) while DNT showed no morphological effects on HEp-2 cells but did induce binucleation of Swiss 3T3 cells. To determine if the enzymatic domain of each toxin dictated the induced phenotype, we constructed enzymatically active chimeric toxins and mutant toxins that contained single amino acid substitutions within the catalytic site and tested these molecules in tissue culture and enzymatic assays. Moreover, both site-directed mutant toxins showed reduced time to maximum transglutamination of RhoA compared with the parent toxins. Nevertheless, the substitution of threonine for Lys(1310) in the DNT-based mutant, while affecting transglutamination efficiency of the toxin, did not abrogate that enzymatic activity.     

Exchange of a single amino acid switches the substrate properties of RhoA and RhoD toward glucosylating and transglutaminating toxins

Rho GTPases are the preferred targets of various bacterial cytotoxins, including Clostridium difficile toxins A and B, Clostridium sordellii lethal toxin, the cytotoxic necrotizing factors (CNF1) from Escherichia coli, and the dermonecrotizing toxin (DNT) from Bordetella species. The toxins inactivate or activate specific sets of Rho GTPases by mono-O-glucosylation and deamidation/transglutamination, respectively. Here we studied the structural basis of the recognition of RhoA, which is modified by toxin B, CNF1, and DNT, in comparison with RhoD, which is solely a substrate for lethal toxin. We found that a single amino acid residue in RhoA and RhoD defines the substrate specificity for toxin B and lethal toxin. Change of serine 73 to phenylalanine in RhoA turned RhoA into a substrate for lethal toxin. Accordingly, change of the equivalently positioned phenylalanine 85 in RhoD with serine allowed glucosylation by toxin B. Comparable results were achieved with the Rho-activating and transglutaminating enzymes CNF1 and DNT. Here, amino acid glutamate 64 of RhoA and the equivalent aspartate 76 of RhoD define substrate specificity for CNF1 and DNT, respectively. These data indicate that single amino acid residues located in the switch II region of Rho proteins determine enzyme specificity for diverse bacterial toxins.     

Bordetella dermonecrotic toxin binds to target cells via the N-terminal 30 amino acids

Bordetella dermonecrotic toxin (DNT) affects the biological function of host cells by activating intracellular Rho GTPases. The toxin binds to unidentified receptor(s) via 54 N-terminal amino acids, undergoes intramolecular cleavage on the C-terminal side of Arg(44) by furin or furin-like protease, and eventually enters the cytoplasm where the Rho GTPases reside. The binding to the receptor(s) and intramolecular cleavage are essential for DNT to intoxicate cells, and the 54 amino-acid binding domain encompasses the cleavage site, however, it is unclear whether these two events are related. In this study, we could narrow down the cell-binding domain to the N-terminal amino acids 2-30. The region does not contain the furin-recognition site, indicating that the cell binding and the intramolecular cleavage are independent events.     

Bordetella dermonecrotic toxin undergoes proteolytic processing to be translocated from a dynamin-related endosome into the cytoplasm in an acidification-independent manner

Bordetella pertussis dermonecrotic toxin (DNT), which activates intracellular Rho GTPases, is a single chain polypeptide composed of an N-terminal receptor-binding domain and a C-terminal enzymatic domain. We found that DNT was cleaved by furin, a mammalian endoprotease, on the C-terminal side of Arg(44), which generates an N-terminal fragment almost corresponding to the receptor-binding domain and a C-terminal remainder (deltaB) containing the enzymatic domain. These two fragments remained associated even after the cleavage and made a nicked form. DNT mutants insensitive to furin had no cellular effect, whereas the nicked toxin was much more potent than the intact form, indicating that the nicking by furin was a prerequisite for action. DeltaB, but not the nicked toxin, associated with artificial liposomes and activated Rho in cells resistant to DNT because of a lack of surface receptor. These results imply that deltaB, dissociated from the binding domain, fully possesses the ability to enter the cytoplasm across the lipid bilayer membrane. The translocation ability of deltaB was found to be attributable to the N-terminal region encompassing amino acids 45-166, including a putative transmembrane domain. Pharmacological analyses with various reagents disturbing vesicular trafficking revealed that the translocation requires neither the acidification of the endosomes nor retrograde vesicular transport to deeper organelles, although DNT appeared to be internalized via a dynamin-dependent endocytosis. We conclude that DNT binds to its receptor and is internalized into endosomes where the proteolytic processing occurs. DeltaB, liberated from the binding domain after the processing, begins to translocate the enzymatic domain into the cytoplasm.     

Modification of plant Rac/Rop GTPase signalling using bacterial toxin transgenes

Bacterial protein toxins which modify Rho GTPase are useful for the analysis of Rho signalling in animal cells, but these toxins cannot be taken up by plant cells. We demonstrate in vitro deamidation of Arabidopsis Rop4 by Escherichia coli Cytotoxic Necrotizing Factor 1 (CNF1) and glucosylation by Clostridium difficile toxin B. Expression of the catalytic domain of CNF1 caused modification and activation of co‐expressed Arabidopsis Rop4 GTPase in tobacco leaves, resulting in hypersensitive‐like cell death. By contrast, the catalytic domain of toxin B modified and inactivated co‐expressed constitutively active Rop4, blocking the hypersensitive response caused by over‐expression of active Rops. In transgenic Arabidopsis, both CNF1 and toxin B inhibited Rop‐dependent polar morphogenesis of leaf epidermal cells. Toxin B expression also inhibited Rop‐dependent morphogenesis of root hairs and trichome branching, and resulted in root meristem enlargement and dwarf growth. Our results show that CNF1 and toxin B transgenes are effective tools in Rop GTPase signalling studies.     

Cytotoxic necrotizing factor 1 fromEscherichia coli: a toxin with a new intracellular activity for eukaryotic cells

Certain pathogenicEscherichia coli strains elaborate a toxin, the cytotoxic necrotizing factor type 1 (CNF1). CNF1 covalently and specifically modifies the p21 Rho GTP-binding protein in mammalian cells by deamidation of the p21 Rho glutamine 63. CNF1 modification of Rho leads to permanent activation of the GTP-binding protein by blocking intrinsic and RhoGAP GTPase activities. Rho activation by CNF1 induces reorganization of the actin cytoskeleton into large stress fibers and the multiplication of focal contact points. Deamidation is a new catalytic activity described for an intracellularly acting toxin. Presented at the1st International Minisymposium on Cellular Microbiology: Cell Biology and Signalization in Host-Pathogen Interactions, Prague, October 6, 1997.     

His1205 and His1223 are essential for the activity of the mitogenic Pasteurella multocida toxin

Pasteurella multocida produces a 146-kDa protein toxin (PMT), which activates multiple cellular signal-transduction pathways, resulting in the activation of PLCbeta, Rho, JNK, and ERK. In addition to an essential cysteine residue at position 1165, PMT contains several histidine residues in the catalytically important C-terminal part of the protein. To elucidate the role of the histidine residues, we treated PMT with the histidine-modifying substance diethyl pyrocarbonate (DEPC). DEPC inhibited PMT in a time- and concentration-dependent manner, suggesting that one or several histidine residues are essential for the biological activity of PMT. In experiments in which PMT was directly delivered into the cytosol of EBL cells by electroporation, we show that DEPC treatment inhibits the catalytically important histidine residues. Leucine substitutions of eight individual histidine residues in the C-terminal catalytic domain of PMT were constructed, and the effect on the biological activity of PMT was analyzed by determining PLCbeta, Rho, and ERK activation. Substitution of two histidine residues, H1205 and H1223, led to inactivation of the resulting PMT proteins, indicating that H1205 and H1223 play an important role in biological activity of the toxin. In addition, we show that the mutant toxins appear to be correctly folded, as judged by protease digestion. The precise function of H1205 and H1223 is not yet known. However, treatment of PMT with the cation chelating substance 1,10-phenantroline led to inactivation of the toxin, indicating that the essential histidine residues and cysteine 1165 might be involved in metal ion binding.     

Activation and proteasomal degradation of rho GTPases by cytotoxic necrotizing factor-1 elicit a controlled inflammatory response

The CNF1 toxin is produced by uropathogenic and meningitis-causing Escherichia coli. CNF1 penetrates autonomously into cells and confers phagocytic properties to epithelial and endothelial cells. CNF1 acts at the molecular level by constitutively activating Rho GTPases attenuated by their cellular ubiquitin-mediated proteasomal degradation. Here we report the relationship between the ubiquitin-mediated proteasomal degradation of activated Rho and the endothelial cell response to the toxin. The type of cellular response to CNF1 intoxication, first screened by DNA microarray analysis, revealed the launching of a program oriented toward an inflammatory response. Parallel to Rho protein activation by CNF1, we also established the kinetics of production of monocyte chemotactic protein-1 (MCP-1), interleukin-8 (IL-8), IL-6, monocyte inflammatory protein-3alpha (MIP-3alpha) and E-selectin. Both the mutation of the catalytic domain of the toxin (CNF1-C866S) and the inhibition of Rho proteins abrogate the CNF1-induced production of the immunomodulators MIP-3alpha, MCP-1, and IL-8. These immunomodulators are also produced upon activation of Cdc42 and Rac preferentially. Our results indicate that, in addition to pathogen molecular pattern recognition by host-receptors, a direct activation of Rho proteins by the CNF1 virulence factor efficiently triggers a cellular reaction of host alert. Consistently, we assume that the CNF1-induced ubiquitin-mediated proteasomal degradation of activated Rho proteins may limit the amplitude of the host cell immune responses.     

37-kDa laminin receptor precursor modulates cytotoxic necrotizing factor 1-mediated RhoA activation and bacterial uptake

Cytotoxic necrotizing factor 1 (CNF1) is a bacterial toxin known to activate Rho GTPases and induce host cell cytoskeleton rearrangements. The constitutive activation of Rho GTPases by CNF1 is shown to enhance bacterial uptake in epithelial cells and human brain microvascular endothelial cells. However, it is unknown how exogenous CNF1 exhibits such phenotypes in eukaryotic cells. Here, we identified 37-kDa laminin receptor precursor (LRP) as the receptor for CNF1 from screening the cDNA library of human brain microvascular endothelial cells by the yeast two-hybrid system using the N-terminal domain of CNF1 as bait. CNF1-mediated RhoA activation and bacterial uptake were inhibited by exogenous LRP or LRP antisense oligodeoxynucleotides, whereas they were increased in LRP-overexpressing cells. These findings indicate that the CNF1 interaction with LRP is the initial step required for CNF1-mediated RhoA activation and bacterial uptake in eukaryotic cells.     

Rho GTPases as targets of bacterial protein toxins

Several bacterial toxins target Rho GTPases, which constitute molecular switches in several signaling processes and master regulators of the actin cytoskeleton. The biological activities of Rho GTPases are blocked by C3-like transferases, which ADP-ribosylate Rho at Asn41, but not Rac or Cdc42. Large clostridial cytotoxins (e. g., Clostridium difficile toxin A and B) glucosylate Rho GTPases at Thr37 (Rho) or Thr35 (Rac/Cdc42), thereby inhibiting Rho functions by preventing effector coupling. The 'injected' toxins ExoS, YopE and SptP from Pseudomonas aeruginosa, Yersinia and Salmonella ssp., respectively, which are transferred into the eukaryotic target cells by the type-III secretion system, inhibit Rho functions by acting as Rho GAP proteins. Rho GTPases are activated by the cytotoxic necrotizing factors CNF1 and CNF2 from Escherichia coli and by the dermonecrotizing toxin DNT from B. bronchiseptica. These toxins deamidate/transglutaminate Gln63 of Rho to block the intrinsic and GAP-stimulated GTP hydrolysis, thereby constitutively activating the GTPases. Rho GTPases are also activated by SopE, a type-III system injected protein from Salmonella ssp., that acts as a GEF protein.     

Activation of Rho GTPases by Cytotoxic Necrotizing Factor 1 Induces Macropinocytosis and Scavenging Activity in Epithelial Cells

Macropinocytosis, a ruffling-driven process that allows the capture of large material, is an essential aspect of normal cell function. It can be either constitutive, as in professional phagocytes where it ends with the digestion of captured material, or induced, as in epithelial cells stimulated by growth factors. In this case, the internalized material recycles back to the cell surface. We herein show that activation of Rho GTPases by a bacterial protein toxin, the Escherichia coli cytotoxic necrotizing factor 1 (CNF1), allowed epithelial cells to engulf and digest apoptotic cells in a manner similar to that of professional phagocytes. In particular, we have demonstrated that 1) the activation of all Rho, Rac, and Cdc42 by CNF1 was essential for the capture and internalization of apoptotic cells; and 2) such activation allowed the discharge of macropinosomal content into Rab7 and lysosomal associated membrane protein-1 acidic lysosomal vesicles where the ingested particles underwent degradation. Taken together, these findings indicate that CNF1-induced "switching on" of Rho GTPases may induce in epithelial cells a scavenging activity, comparable to that exerted by professional phagocytes. The activation of such activity in epithelial cells may be relevant, in mucosal tissues, in supporting or integrating the scavenging activity of resident macrophages.     

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.     

Cellular uptake of Clostridium difficile toxin B. Translocation of the N-terminal catalytic domain into the cytosol of eukaryotic cells

Clostridium difficile toxin B (269 kDa) is one of the causative agents of antibiotic-associated diarrhea and pseudomembranous colitis. Toxin B acts in the cytosol of eukaryotic target cells where it inactivates Rho GTPases by monoglucosylation. The catalytic domain of toxin B is located at the N terminus (amino acid residues 1-546). The C-terminal and the middle region of the toxin seem to be involved in receptor binding and translocation. Here we studied whether the full-length toxin or only a part of the holotoxin is translocated into the cytosol. Vero cells were treated with recombinant glutathione S-transferase-toxin B, and thereafter, toxin B fragments were isolated by affinity precipitation of the glutathione S-transferase-tagged protein from the cytosolic fraction of intoxicated cells. The toxin fragment (approximately 65 kDa) was recognized by an antibody against the N terminus of toxin B and was identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis as the catalytic domain of toxin B. The toxin fragment located in the cytosol possessed glucosyltransferase activity that could modify RhoA in vitro, but it was not able to intoxicate intact cells. After treatment of Vero cells with a radiolabeled fragment of toxin B (amino acid residues 547-2366), radioactivity was identified in the membrane fraction of Vero cells but not in the cytosolic fraction of Vero cells. Furthermore, analysis of cells by fluorescence microscopy revealed that the C terminus of toxin B was located in endosomes, whereas the N terminus was detected in the cytosol. Protease inhibitors, which were added to the cell medium, delayed intoxication of cells by toxin B and pH-dependent translocation of the toxin from the cell surface across the cell membrane. The data indicate that toxin B is proteolytically processed during its cellular uptake process.     

Rho GTPase-activating bacterial toxins: from bacterial virulence regulation to eukaryotic cell biology

Studies on the interactions of bacterial pathogens with their host have provided an invaluable source of information on the major functions of eukaryotic and prokaryotic cell biology. In addition, this expanding field of research, known as cellular microbiology, has revealed fascinating examples of trans-kingdom functional interplay. Bacterial factors actually exploit eukaryotic cell machineries using refined molecular strategies to promote invasion and proliferation within their host. Here, we review a family of bacterial toxins that modulate their activity in eukaryotic cells by activating Rho GTPases and exploiting the ubiquitin/proteasome machineries. This family, found in human and animal pathogenic Gram-negative bacteria, encompasses the cytotoxic necrotizing factors (CNFs) from Escherichia coli and Yersinia species as well as dermonecrotic toxins from Bordetella species. We survey the genetics, biochemistry, molecular and cellular biology of these bacterial factors from the standpoint of the CNF1 toxin, the paradigm of Rho GTPase-activating toxins produced by urinary tract infections causing pathogenic Escherichia coli. Because it reveals important connections between bacterial invasion and the host inflammatory response, the mode of action of CNF1 and its related Rho GTPase-targetting toxins addresses major issues of basic and medical research and constitutes a privileged experimental model for host-pathogen interaction.     

Activation of rho through a cross-link with polyamines catalyzed by Bordetella dermonecrotizing toxin

The small GTPase Rho, which regulates a variety of cell functions, also serves as a specific substrate for bacterial toxins. Here we demonstrate that Bordetella dermonecrotizing toxin (DNT) catalyzes cross-linking of Rho with ubiquitous polyamines such as putrescine, spermidine and spermine. Mass spectrometric analyses revealed that the cross-link occurred at Gln63, which had been reported to be deamidated by DNT in the absence of polyamines. Rac1 and Cdc42, other members of the Rho family GTPases, were also polyaminated by DNT. The polyamination, like the deamidation, markedly reduced the GTPase activity of Rho without affecting its GTP-binding activity, indicating that polyaminated Rho behaves as a constitutively active analog. Moreover, polyamine-linked Rho, even in the GDP-bound form, associated more effectively with its effector ROCK than deamidated Rho in the GTP-bound form and, when microinjected into cells, induced the anomalous formation of stress fibers indistinguishable from those seen in DNT-treated cells. The results imply that the polyamine-linked Rho, transducing signals to downstream ROCK in a novel GTP-independent manner, plays an important role in DNT cell toxicity.     

Cytotoxic necrotizing factor 1 prevents apoptosis via the Akt/IkappaB kinase pathway: role of nuclear factor-kappaB and Bcl-2

Cytotoxic necrotizing factor 1 (CNF1) is a protein toxin produced by some pathogenic strains of Escherichia coli that specifically activates Rho, Rac, and Cdc42 GTPases. We previously reported that this toxin prevents the ultraviolet-B-induced apoptosis in epithelial cells, with a mechanism that remained to be defined. In this work, we show that the proteasomal degradation of the Rho GTPase is necessary to achieve cell death protection, because inhibition of Rho degradation abolishes the prosurvival activity of CNF1. We hypothesize that Rho inactivation allows the activity of Rac to become dominant. This in turn leads to stimulation of the phosphoinositide 3-kinase/Akt/IkappaB kinase/nuclear factor-kappaB prosurvival pathway and to a remarkable modification in the architecture of the mitochondrial network, mainly consisting in the appearance of elongated and interconnected mitochondria. Importantly, we found that Bcl-2 silencing reduces the ability of CNF1 to protect cells against apoptosis and that it also prevents the CNF1-induced mitochondrial changes. It is worth noting that the ability of a bacterial toxin to induce such a remodeling of the mitochondrial network is herein reported for the first time. The possible pathophysiological relevance of this finding is discussed.     

CNF1 exploits the ubiquitin-proteasome machinery to restrict Rho GTPase activation for bacterial host cell invasion

CNF1 toxin is a virulence factor produced by uropathogenic Escherichia coli. Upon cell binding and introduction into the cytosol, CNF1 deamidates glutamine 63 of RhoA (or 61 of Rac and Cdc42), rendering constitutively active these GTPases. Unexpectedly, we measured in bladder cells a transient CNF1-induced activation of Rho GTPases, maximal for Rac. Deactivation of Rac correlated with the increased susceptibility of its deamidated form to ubiquitin/proteasome-mediated degradation. Sensitivity to ubiquitylation could be generalized to other permanent-activated forms of Rac and to its sustained activation by Dbl. Degradation of the toxin-activated Rac allowed both host cell motility and efficient cell invasion by uropathogenic bacteria. CNF1 toxicity thus results from a restricted activation of Rho GTPases through hijacking the host cell proteasomal machinery.     

Identification and characterization of the Pasteurella multocida toxin translocation domain

The Pasteurella multocida toxin (PMT) is a potent mitogen which enters the cytosol of eukaryotic cells via a low pH membrane translocation event. In common with the Escherichia coli cytotoxic necrotizing factor 1 (CNF1), the core of the PMT translocation domain is composed of two predicted hydrophobic helices (H1 - residues 402-423, H2 - 437-457) linked by a hydrophilic loop (PMT-TL - 424-436). The peptide loop contains three acidic residues (D425, D431 and E434), which may play a role equivalent to D373, D379 and E382/383 in CNF1. To test this hypothesis, a series of point mutants was generated in which acidic residues were mutated into the permanently charged positive residue lysine. Individual mutation of D425, D431 and E434 each caused a four- to sixfold reduction in toxin activity. Interestingly, mutation of D401 located immediately outside the predicted helix-loop-helix motif completely abolished toxin activity. Individual mutations did not affect cell binding nor greatly altered toxin structure, but did prevent translocation of the surface-bound proteins into the cytosol after a low pH pulse. Moreover, we demonstrate using an in vitro assay that PMT undergoes a pH-dependent membrane insertion.