Pore-forming Peptides and Protein Toxins (Cellular and Molecular Mechanisms of Toxic Action)

Special Issue "Pore-Forming Toxins"

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Of 11 dysfunctional mutations marked by asterisk in Fig. This suggests that the most important part of the protein, responsible for toxicity and lipid binding, comprises the loops at the top of the molecule as oriented in Fig. While bearing in mind that there are no data on the possible effect of the mutations on the structural stability of the toxin itself, one may tentatively assume that the mutated residues are important for electrostatic interactions with polar head groups of lipids.

With more new mutants, it might be possible to probe the role of selected hydrophobic residues. Alternatively, cysteine residues inserted at judiciously selected locations might allow for disulfide clamping of Cyt1A, which may render whole regions of the toxin dimer surface unavailable for interaction with membranes. The latter approach would be especially informative in terms of pinpointing the regions responsible for lipid binding.

After their initial observation that dissolved B. These authors demonstrated that the determinants important for toxin binding were the nature of the lipid polar head group phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin bound the toxin and the presence of unsaturated fatty acyl chains. Haider and Ellar 22 found that the addition of B. In fact, visualization or sizing of lipid assemblies or lipid-toxin complexes after the lytic action of Cyt1A might be the crucial contribution to determining the toxin's mode of action, as discussed in more detail below.

Unfortunately, these authors did not record single-channel conductance but had to rely on multiple-channel recordings. Consequently, discrete conductance levels are not easily discernible from their data.

Moreover, determining whether discrete conductance steps are due to formation of channels or to a much less specific perturbation of bilayer structure mediated by proteins is not always straightforward. A more thorough analysis of single-channel recordings of Cyt2Aa1, a toxin from B.

This work proved unequivocally that potassium channels are observed for at least 10 min after incorporation of the activated toxin into planar lipid bilayers. These authors also observed that upon incorporation into liposomes with entrapped glucose, the toxin caused glucose release. This raises the interesting question of whether glucose passes through a cation-selective channel or the toxin forms different kinds of openings in membranes of different radii of curvature.

All of the papers mentioned so far 22 , 24 , 26 , 42 , 43 have been cited in support of the colloid-osmotic lysis mechanism of Cyt1A-mediated cell damage According to this hypothesis, Cyt1A forms cation-selective channels in the cell membrane; equilibration of cation concentrations across the membrane results in osmotic movement of water with subsequent swelling and eventual rupture of the cell. In the absence of direct physical evidence for a proteinaceous channel or pore, several research groups investigated the interaction between Cyt1A and lipids with the aim of identifying the protein regions that embed in the lipid bilayer.

They synthesized the peptide and determined that it was hemolytic.

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Gazit and Shai 19 fluorescently labeled two putative hydrophobic segments amino acids to and amino acids 50 to 71 and used them in lipid binding assays. Both peptides bound strongly to zwitterionic lipids, self-associated, and interacted with each other; the segment consisting of amino acids to permeabilized lipid vesicles.

Do these two studies constitute the proof that Cyt1A inserts in the membrane and forms proteinaceous channels or pores? The structural predictions of Szabo et al. However, a synthetic-peptide approach to studying mechanisms of protein-lipid interactions may not always be the best: For example, circular dichroism spectra of the synthetic segment comprising amino acids to showed that this peptide is helical in trifluoroethanol but not in water Furthermore, extrinsic fluorescence labeling may alter physicochemical properties of the peptides, most notably their partitioning in the membrane.

Gazit and Shai 19 reported that the probe with N-terminal fluorescence probe was localized in the hydrophobic interior of the bilayer. This fact by itself rules out the possibility that the helical peptides span the membrane: Part of this criticism has already been formulated 4.

In , Li et al. The strands also appear to be protected from attack by water-soluble proteases, which suggests that they may span the bilayer rather than lie parallel to its surface. A comprehensive synthesis of the original seminal observations from the s 29 , 43 , 45 and newer data 11 , 28 , 33 with preliminary results obtained by novel techniques such as acoustic waveguide spectroscopy and atomic force microscopy was recently presented by Ellar A working model of the putative Cyt pore consists of six toxin molecules assembled like an open umbrella.

By definition, transmembrane pores or channels must contain at least one part of the molecule that reaches across the lipid bilayer to the other side of the membrane. It is noteworthy that Du et al. The most obvious explanation of this negative result would be that no part of the toxin penetrates the interior surface of the membrane.

A previous fluorescence spectroscopy study 4 probed the localization of the tryptophan region strand 4 and the flanking areas with respect to the membrane surface. The observed lack of a blue shift, lack of quenching by lipidic quenchers, and decreased quantum yield in the presence of lipid all suggested that the region of Cyt1A where the two tryptophans are located is not inserted in the lipid bilayer. However, this does not clearly challenge the pore hypothesis since these experiments did not address the localization of other parts of the protein, which were invisible to the spectrofluorometer.

Several other data do question the notion of well-defined protein-lined pores formed by Cyt1A. First, the Cyt1A-induced leakage from vesicles exhibited kinetics independent of the size of the leaking molecule, between 0. This would be unlikely if the diameter of the pore were well defined as 1 or 2 nm This indicates that when the toxin is bound to lipid, all the amide protons are easily accessible to the aqueous solvent, suggesting that no significant part of Cyt1A inserts in the lipid bilayer core.

Cyt1A is mostly or perhaps completely splayed out on the membrane surface. This hypothesis is consistent with another recent finding of Du et al.

Of course, these results are also consistent with the umbrella-like pore model. Additional biophysical data 4 lend further support to this hypothesis: Thus, it is only the tertiary structure that is loosened upon lipid binding; the bulk of the secondary structure is retained, perhaps in a form similar to the so-called molten-globule state The only question pertaining to these experiments is whether the sensitivity of the employed biophysical methods is sufficient to detect a few amides out of the peptide bonds in Cyt1A that might be buried in the membrane.

Aggregation of Cyt1A in the membrane appears to play an important role in the pore hypothesis. When a transmembrane pore is formed, often several molecules of toxin assemble to create the lining of the pore, as is the case with, e. From a concentration-dependent lag of the toxin effect on the Malpighian tubules of insects 29 and from the analysis of Cyt1A-mediated release of a fluorescent dye from large unilamellar lipid vesicles 3 , it was concluded that the membrane is affected only after it has harbored a sufficiently large number of Cyt1A molecules.

Nevertheless, there is only one published report of direct physical isolation, by sucrose density gradient centrifugation, of Cyt1A aggregates: One drawback of this study is the uncertain composition of the aggregates due to the use of whole cells, whose membranes contain endogenous proteins. Cyt1A might be complexed with those proteins rather than with itself. This caveat notwithstanding, there is now little disagreement on the notion that several and perhaps many molecules of Cyt1A must come into contact in the membrane for cytolysis to occur.

As the channels or pores are usually formed by assembling a small number of protein subunits with a more or less constant stoichiometry, the pore hypothesis would be supported by finding only small oligomers. If, on the other hand, no aggregates, or only large aggregates, are found, an alternative hypothesis must be considered. This issue was directly addressed by Promdonkoy and Ellar 32 ; Promdonkoy and Ellar, unpublished.

In the presence of the membrane, Cyt2A exhibited a laddering pattern in sodium dodecyl sulfate-polyacrylamide gel electrophoresis when the samples were not boiled before electrophoresis. However, the ladder did not show a molecular size limit or the maximum staining intensity in the region of to kDa, which would be expected for hexameric stoichiometry.

Pore-forming Peptides and Protein Toxins

Aggregates so large that they could not enter the gel were apparent in some, but not all, blots. The authors unsuccessfully attempted to electrophorese pore complexes stabilized by glutaraldehyde cross-linking. Perhaps a more specific distance-sensitive protein cross-linker that will covalently link only closely opposed proteins in the membrane of a lipid vesicle could be used to form stable higher-molecular-weight aggregates of the toxin, which could then be unequivocally resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

They have divergent sequences, and are classified by Pfam into a number of families including Leukocidins, Etx-Mtx2, Toxin, and aegerolysin. X-ray crystallographic structures have revealed some commonalities: Similarly, aerolysin [5] and Clostridial Epsilon-toxin. These toxins are potent but also highly specific to a limited range of target insects, making them safe biological control agents.

The partner proteins in these combinations may belong to different structural groups, depending on the individual toxin: The 'cap' of the mushroom sits on the surface of the cell, and the 'stalk' of the mushroom penetrates the cell membrane, rendering it permeable see later. A structure of the Vibrio cholerae cytolysin [14] in the pore form is also heptameric; however, Staphylococcus aureus gamma-hemolysin [15] reveals an octomeric pore, consequently with a strand 'stalk'. The Panton-Valentine leucocidin S structure [16] shows a highly related structure, but in its soluble monomeric state.

Pore-forming toxin

This shows that the strands involved in forming the 'stalk' are in a very different conformation — shown in Fig 2. A structure of the Vibrio cholerae cytolysin PDB: While the Bin toxin of Lysinibacillus sphaericus is able to form pores in artificial membranes [20] and mosquito cells in culture, [21] it also causes a series of other cellular changes including the uptake of toxin in recycling endosomes and the production of large, autophagic vesicles [22] and the ultimate cause of cell death may be apoptotic.

The transition between soluble monomer and membrane-associated protomer to oligomer is not a trivial one: Following this, the large-scale conformational change occurs in which the membrane spanning section is formed and inserted into the membrane. The portion entering the membrane, referred to as the head, is usually apolar and hydrophobic, this produces an energetically favorable insertion of the pore-forming toxin.

Ions and small molecules, such as amino acids and nucleotides within the cell, flow out, and water from the surrounding tissue enters. The loss of important small molecules to the cell can disrupt protein synthesis and other crucial cellular reactions. The loss of ions, especially calcium , can cause cell signaling pathways to be spuriously activated or deactivated. The uncontrolled entry of water into a cell can cause the cell to swell up uncontrollably: In the end, this can cause the cell to burst.

There are many different types of binary toxins. The term binary toxin simply implies a two part toxin where both components are necessary for toxic activity. The interplay of the individual components has not been well studied to date. Other beta sheet toxins of commercial importance are also binary. The DAF-2 network includes a putative E3 ubiquitin ligase, WWP-1, involved in promoting innate immunity against pathogenic bacteria and in promoting life span [ 9 ]. Mutations in wwp -1 gene become hypersensitive six fold to Cry5B toxin, suggesting that this protein is involved in the nematode defense to PFT [ 9 ].

AKT is also known as protein kinase B or PKB, it is a key regulator of host cell survival playing an important role in controlling cell cycle, vesicular trafficking, apoptosis inhibition, and inflammatory responses to bacterial infections. This inactivation of AKT by PFT adversely affects host inflammatory responses and induces apoptotic response at sublytic concentrations [ 42 ]. The activated SREBP function as transcription factor that promoted the synthesis of genes involved in membrane biogenesis, leading into a defense response against to PFT damage. Pore formation activity is linked to this response since toxin mutants affected in membrane insertion or in oligomer formation that lack pore formation activity did not activate SREBP pathway [ 20 ].

The analysis of cellular responses to PFT has revealed conserved responses in different organisms. Among these, activation of protein kinase p38 is a conserved response in nematodes, insects and mammals. This signal transduction pathway is triggered by low doses of PFT promoting in most of the cases the cell survival. Different isoforms of p38 kinases a, b, g and d have been identified in mammalian cells [ 33 ], and each one of these proteins has specific functions determined by their upstream activators and their downstream substrates resulting in initiation of a wide variety of secondary responses.

The secondary consequences of p38 activation will depend on the type of PFT and on the target cell, resulting in initiation of different mechanisms involved in protection against PFT, and in few cases in activation of programmed death responses. The identification of the different scaffold proteins that are responsible of the selective and accurate responses activated by p38 after PFT intoxication in different organisms could provide ways to counter act the action of bacterial pathogens or to enhance the activity of biotechnological important PFT as Cry toxins produced B.

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Abstract Understanding the mechanism of action of pore-forming toxins PFT produced by different bacteria, as well as the host responses to toxin action, would provide ways to deal with these pathogenic bacteria. Introduction Pore-forming toxins PFT produced by bacteria are virulence factors which play a fundamental role in killing eukaryotic cells by forming holes in the cellular membrane of their targets.

Open in a separate window. Downstream targets of MAPK p38 pathway specifically activated in response to PFT Regarding the identification of the key downstream targets of the p38 pathway that are specifically activated in response to PFTs, it was shown by comparison of microarray analysis of C. Other bacterial toxins non-PFT that affect MAPK p38 pathway The shiga toxin STx is a member of the AB5 family which is characterized by a bipartite structure consisting of a pentameric B5 moiety involved in receptor binding and the catalytic A subunit, which is a N-glycosidase that inactivates the protein synthesis machinery.

Final Remarks The analysis of cellular responses to PFT has revealed conserved responses in different organisms. Mannehemia haemolytica leokotoxin induces apoptosis of bovine lymphoblastoid cells BL-3 via caspase-9 dependent mitochondrial pathway. Hypoxia and the hypoxic response pathway protect against pore-forming toxins in C. Adenylate cyclase toxin CyaA of Bordetella pertrussis. Evidence for the formation of small ion permeable channels and comparison with HlyA of Escherichia coli. Activation of the unfolded protein response is required for defenses against bacterial pore-forming toxin in vivo.

The mitogen-activated protein kinase p38 is involved in insect defense against Cry toxins from Bacillus thuringiensis.

Insect Biochem Mol Biol. Defense and death responses to pore forming toxins. Biotechnol Gen Eng Rev.

Chang L, Karin M. Mammalian MAP kinase signalling cascades. WWP-1 is a novel modulator of the DAF-2 insulin-like signaling network involved in pore-forming toxin cellular defenses in Caenorhabditis elegans. Nuclear localization and regulation of Erk- and Rsk-encoded protein kinases. Anthrax lethal toxin promotes dephosphorylation of TTP and formation of processing bodies.

MAP kinases in the immune response. Dynamics and organization of MAP kinase signal pathways. Functional and phylogenetic characterization of vaginolysin the human specific cytolysin from Gardnerella vaginalis. Streptococcus pneumoniae R6x induced p38 and JNK-mediated caspase-dependent apoptosis in human endothelial cells. Caspase-1 activation of lipid metabolic pathways in response to bacterial pore-forming toxins promotes cell survival.

Mitogen-activated protein kinase pathways defend against bacterial pore-forming toxins. Pore-forming toxins activate MAPK p38 by causing loss of cellular potassium. Biochem Biophys Res Commun. Nature Rev Mol Cell Biol. Krishna M, Narang H. The complexity of mitogen-activated protein kinases MAPK made simple.

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