MB-JASS 2007 – Session III – Properties of Channels Formed by Bacterial Porins and Toxins MB-JASS 2007 – Session III “Properties of Channels Formed by Bacterial Porins and Toxins” March 11.-21. 2007 – Moscow, Russia “RTX Toxins: HlyA of Escherichia coli & CyaA of Bordetella pertussis.” Florian Rohleder, Oliver Knapp & Roland Benz Department of Biotechnology, University Würzburg, Germany The RTX toxins: RTX-Toxins are important virulence factors produced by a wide range of Gramnegative bacteria (Lally et al, 1999). The RTX-toxin family consists currently of 16 proteins for which the genes have been sequenced (Welch et al, 1995). Examples for important cytolysin of this family are the E. coli -Hemolysin (HlyA), the Actinobacillus pleuropneumoniae ApxI-toxin and the Bordetella pertussis adenylate cyclase toxin. Some RTX-Toxins, like the -Hemolysin, act on a wide range of different host cell types, but others, e. g. the leukotoxins of A. actinomycetemcomitans, act only on a restricted number of cells in a species-specific fashion. Leukotoxin A from A. actinomycetemcomitans kills lymphocytes and granulocytes from humans, the great apes and Old World Monkeys, whereas Leukotoxin A from Pasteurella haemolytica acts only on bovine white blood cells (Lally et al, 1999). RTX (repeats in toxin) toxins are members of a family of proteins that are synthesized by a diverse group of Gram-negative pathogens. All members of the RTX toxin family share a common gene organization and distinctive structural features. Although variations do exist, the generic RTX toxin operon consists of four different genes that are designated rtxC, A, B and D in transcriptional order. Most RTX toxins require post-translational modification to become biologically active. The MB-JASS 2007 – Session III – Properties of Channels Formed by Bacterial Porins and Toxins toxins are transported from the cytoplasm to the cell surface by transport proteins encoded by the rtxB and rtxD genes. RtxA contains tandemly repeated nonapeptides that have the consensus sequence UXGGXG(N/D)DX, with U standing for large hydrophobic and X for any given amino acid. The number of times this motif is repeated varies, ranging from six to 40 with individual toxins, but the presence per se defines this group of toxins. RtxB proteins are members of the ATP-binding cassette superfamily of transport proteins and rtxD proteins, which are unique to prokaryotes, belong to the membrane fusion protein family (Lally et al, 1999). Calcium binding by these regions is absolutely necessary for biological activity. In the N-terminal sequences occurs the greatest sequence divergence among the different toxins (Welch, 2001). HlyA of E. coli: Escherichia coli haemolysin (HlyA) is a major cause of E. coli virulence and E. coli αhemolysin (HlyA) is the best-characterized RTX protein secreted by a type I secretion system. It is mainly produced by E. coli strains causing urinary tract infections (uropathogenic E. coli; UPEC) and is an important virulence factor owing to its cytolytic and cytotoxic activity against a wide range of mammalian cell types (e.g. erythrocytes, granulocytes, monocytes and endothelial cells). The synthesis, activation and secretion of E. coli HlyA are determined by the hlyCABD operon (Gentschev et al, 2002). The channels formed by the haemolysin can be directly demonstrated also in purely lipidic model systems such as planar membranes and unilamellar vesicles, which lack any putative protein receptor. HlyA has been recognised as a member of a large family of exotoxins elaborated by Gram-negative organisms including Proteus, Bordetella, Morganella, Pasteurella and Actinobacillus. These toxins have quite different target cell specificity and in many cases are leukocidal. When tried on planar membranes however, even specific leukotoxins open channels not dissimilar from those formed by HlyA, suggesting this might be a common step in their action (Menestrina et al, 1994). The operon of HlyA from E. coli consists of four different genes (hlyC, hlyA, hlyB, hlyD), arranged in the named order and comparable to the operon organisation of other RTX toxins. In E. coli, this MB-JASS 2007 – Session III – Properties of Channels Formed by Bacterial Porins and Toxins operon is located either on chromosome-bound pathogenicity islands or on transmissible plasmids. HlyC is the activator protein and HlyB and HlyD form the ABC protein and MFP component of the ABC exporter of HlyA. The outer membrane component of the exporter is TolC which also belongs to the multi-drug efflux-pump and type I secretion system (Andersen, 2003). The crystal structure of TolC shows that its trimeric state forms a trans-periplasmic channel-tunnel with an internal diameter of 35 Å and is about 140 Å in length, comprising a 40 Å-long outer membrane β-barrel (the channel domain) anchoring a contiguous 100 Å-long αhelical barrel that projects across the periplasmic space (the tunnel domain) (Koronakis et al, 2000). Single channel recordings of HlyA of E. coli in artificial bilayers composed of asolectin/n-decane reveal transient channels with a lifetime of 2-5 seconds and a single channel conductance of 1500 pS in 1 M KCl. Experiments with different salts suggested that the haemolysin channel was highly cation-selective at neutral pH. The mobility sequence of the cations in the channel is similar if not identical to their mobility sequence in the aqueous phase and the single-channel data is consistent with a wide, water-filled channel with an estimated minimal diameter of about 1 nm (Benz et al, 1992). CyaA of B. pertussis: The bifunctional RTX (repeat in toxin) adenylate cyclase (AC) toxin-hemolysin (CyaA, ACT, or AC-Hly) is a key virulence factor of the whooping cough agent Bordetella pertussis. CyaA consists of an N-terminal AC enzyme domain (first 400 residues) and of an ~1,300-residue-long pore-forming RTX haemolysin moiety. The latter mediates cell binding and enables the toxin to deliver its catalytic AC domain into the cytosol, where the AC is activated by calmodulin and catalyzes uncontrolled conversion of cellular ATP to cyclic AMP (cAMP), a key second messenger molecule. Besides that, CyaA can form small cation-selective pores in target cell membranes, which accounts for its moderate hemolytic activity on erythrocytes (Benz et al, 1994). After secretion, receptor binding at the target cell, and insertion into cytoplasmic membrane, the toxin evolves its deadly action. High RTX-toxin concentrations induce a very rapid cell death. By contrast, cells exposed to low concentrations show MB-JASS 2007 – Session III – Properties of Channels Formed by Bacterial Porins and Toxins indications of apoptosis. The reason for this behaviour is not known, till now it seems that mitochondrial dysfunction can be correlated with the amount of applied toxin (Lally et al, 1999). Activation of the precursor toxin is mediated through the action of RtxC, which binds covalently fatty acids to one or two special binding sites (Stanley et al, 1994). The mature toxin is actively secreted from the bacteria by a specific transport system consisting of the gene products RtxB and RtxD, located in cytoplasmic membrane, and an additional outer membrane protein, which is in case of E. coli, TolC (Döbereiner et al, 1996). Normally, the tolC gene is not linked with the operon in the same cell. The only exception represents B. pertussis, with the tolC analog being located on the same operon (Laoide and Ullmann, 1990). The transport mechanism belongs to the type I secretion system and mediates the export in one step across the two bacterial membranes independent of the Sec machinery (Lally et al, 1999; Andersen, 2003). Unfortunately no details are know about the 3-D structure of the RTX-toxin channels. For this reason it must be speculated whether a single molecule or a oligomer is involved in transmembrane pore formation. ACT is essential in the early stage of bacterial colonisation of the respiratory tract (Sebo, 1997; Goodwin and Weiss, 1990). It enables the bacteria to escape the host immune system, by intoxication of macrophages and neutrophils. Such treated cells posses no longer the ability to phagoctose and at least apoptosis is induced (Confer and Eaton, 1982). ACT differs from other RTX-toxins through its assembly and function. The 177kDa protein is a bifunctional toxin that exhibits both adenylate cyclase and cytolytic activity. The first 400 N-terminal amino acids harbour the adenylate cyclase domain, which is translocated through an unknown mechanism into the host cell and needs eukaryotic calmodulin for activation (Ludwig and Göbel, 1999). Within the host cell the cyclase produces supraphysological cAMP level, a signal molecule that hence interrupts cellular functions (Confer and Eaton, 1982). This part shows some homologies to the cyclases edema factor (EF) of Anthrax toxin found in Bacillus anthracis (Collier and Young, 2003) and ExoY secreted from Pseudomonas aeruginosa. Homologies are especially found at calmodulin and ATP binding sites (Yahr et al, 1998). Sequence homologies and the structural isolation of these bacterial adenylate cyclases may suppose that this bifunctional B. pertussis toxin arise by fusion of an adenylate cyclase and RTX-toxin gene. The C-terminus consists of 1306 residues and represents the hemolysin domain of the toxin. Within this part one distinguishes two distinct domains. Between residues MB-JASS 2007 – Session III – Properties of Channels Formed by Bacterial Porins and Toxins 500-700 six hydrophobic α-helical structures with amphiphatic and hydrophobic segments are postulated (Benz et al, 1994). This region accounts for membrane insertion and shows some homology to the pore forming region of other RTX-toxins, e. g. the E. coli α-hemolysin (Ludwig et al, 1987; Sebo, 1997). Furthermore the hydrophobic part is able to form small cation-selective transmembrane channels (Benz et al, 1994) and cause osmotic cell lysis. Deletions within this region (residue 623-780 and 827-887) prevent translocation of the adenylate cyclase into the host cell and reduce the hemeolytic activity of ACT. The lack of the adenylate cyclase (AC) domain (1-373) or the C-terminal nonapeptide rich part (1009-1706) has no influence on the channel properties (Benz et al, 1994; Knapp et al, 2003). Mutations effecting the glutamate at position 509 or 516, which are located in the predicted αhelical transmembrane structure, show a significant altering of the channel properties and the protein translocation. Whereas neutral substitutions have only little effect on the toxin activities, charge exchange by lysine reduces the translocation rate of the catalytic domain as well as the hemolytic activity, ion selectivity and channel forming captivity. A substitution of glutamate 509 by a helix breaking proline abolishes totally the invasion of the AC domain, whereas channel formation and cell binding remains unaffected (Osickova et al, 1999). The next domain contains 38 repeats, which are typical for RTX-toxins and unique by their high number. The repeats are involved in receptor and calcium binding. It has been postulated that each repeat binds a single calcium ion with a binding constant between 0.5-0.8 mM and circular dichroism spectroscopy analysis revealed that calcium binding is associated with a conformational change of CyaA. Other studies showed, that calcium binding induces the formation of five β-sheet helices within the C-terminal domain, which seems to be necessary for cell intoxication (Rhodes et al, 2001). Beneath this low calcium affinity side a high affinity binding side could also be identified, but it was – to date – not possible to estimate a binding constant or to localise the responsible residues with the protein. MB-JASS 2007 – Session III – Properties of Channels Formed by Bacterial Porins and Toxins Structural organization of the adenylate cyclase toxin from Bordetella pertussis. The catalytic domain is divided into the two subdomains T25 and T18. CBS represents the calmodulin binding side and the section I, II, and III are involved in catalysis (Ladant and Ullmann, 1999 ). The toxin precursor requires for activation the post-translational palmitoylation of lysine 983 through the gene product of cyaC (Barry et al, 1991). The activated soluble protein - the main part remains bound to the outer membrane of the bacteria and stays inactive - binds after secretion the αMβ2 integrin (CD11b/CD18) receptor which is found on a wide range of different cell types. After binding and channel formation by the hydrophobic domain, the AC penetrates into the host cell and starts with the calmodulin dependent cAMP production (Confer and Eaton, 1982). The exact mechanism of AC delivery and hemolysis is still not known, and there is some disagreement whether the translocation of the enzymatic component and the channel formation are two distinct mechanisms or if both processes are needed for a successful intoxication. One model assumes that the water soluble toxin exists in two conformational isomers. After membrane insertion one isomer mediates the translocation of the catalytic domain, whereas the other one represents a channel precursor. Hemolysis occur after the oligomerisation of the channel precursor proteins (Osickova et al, 1999). MB-JASS 2007 – Session III – Properties of Channels Formed by Bacterial Porins and Toxins Submillimolar concentrations of calcium ions have a deep impact on the channel forming capacity of CyaA (Knapp et al, 2003). The observed activity enhancement of CyaA due to calcium binding is rather consistent with the in vivo situation, where free calcium is present at millimolar concentrations in plasma and body fluids bathing the surface of CyaA target cells, while typically a very low calcium concentration is present in the cell cytosol (around 100 nM). Particularly noteworthy is the extreme (~50-fold) increase of the channel activity of CyaA upon increase of the calcium concentration within a very narrow range from 0.7 to 0.8 mM, hence by only as little as 15%. This strongly indicates that the toxin molecule undergoes a true conformational switching from the essentially “off” state to the “on” conformation that accounts for its high membrane activity and which occurs at higher than 0.6 –0.8 mM free calcium concentrations. The most plausible interpretation of this toggle-like behavior of CyaA is that in the range of 0.6–0.8 mM concentrations of Ca2+ the binding of calcium ions to CyaA proceeds in a highly cooperative manner and at numerous binding sites concomitantly. Such cooperative calcium binding can be expected to cause a major conformational change and/or even partial refolding of the protein. This might possibly consist of formation of parallel β-roll structures upon calcium binding to the numerous low- affinity calcium binding sites within the RTXrepeats of CyaA, as predicted by analogy to the parallel β-roll motifs of the RTXrepeats of Pseudomonas and Serratia proteases, where calcium is bound within the turns connecting the β-strands (Baumann et al, 1993). In parallel, this conformational change might involve also mutual positioning of the calcium-bound β-sheet blocks within the CyaA molecule, possibly due to formation of helical structures within the loops linking the repeat blocks, as suggested by our earlier results on calciuminduced conformational changes in the CyaA molecule as observed by circular dichroism (CD) spectroscopy. The effect of calcium on membrane activity of CyaA appeared to be highly specific. Other divalent cations, such as Mg2+, Sr2+, and Ba2+, had no or very low effect on formation of CyaA channels, and there was no competition between calcium ions and the other divalent cations. Even at very high (20 mM) concentrations the Mg2+, Sr2+, and Ba2+ cations did not interfere with the enhancement of CyaA-mediated channelforming activity by calcium present at submillimolar concentrations. It is noteworthy in this respect that a similar cation selectivity has been found also for the activity of E. coli HlyA (-hemolysin) on model membranes, although strontium and barium MB-JASS 2007 – Session III – Properties of Channels Formed by Bacterial Porins and Toxins induced some HlyA activity (Ostolaza et al, 1995). In sharp contrast, however, channel formation by HlyA in lipid bilayer membranes did not require the presence of calcium ions, and the channel-forming activity of HlyA remained unaltered upon deletion of the RTX repeats or removal of free calcium ions (Döbereiner et al, 1996; Ludwig et al, 1988; Basler et al, 2007). This represents a remarkable difference between the two RTX toxins, which could be accounted for by the specific structure of the RTX domain of CyaA that contains many more calcium binding repeats (~40), as compared to the 13 repeats found in HlyA (Ludwig et al, 1988). The intact CyaA and the mutant ACT1008, in which 698 amino acids of the repeats were removed, exhibited, indeed, the same small channel-forming ability when no calcium ions were added to the assay system (Benz et al, 1994). At calcium concentrations higher than 0.6 mM, however, the membrane activity of intact CyaA was strongly increased compared to ACT1008, which did not respond to increased calcium concentrations at all (Knapp et al, 2003). This strongly indicates that binding of calcium ions to the repeats of CyaA accounted for the calcium effect on its membrane activity. The AC domain of CyaA is not involved in formation of the membrane channels. No interference of calmodulin binding to the AC domain with the formation of channels by CyaA could be observed, and the removal of the AC domain did not affect channel formation, either (Benz et al, 1994). This provides further support for the recently proposed model suggesting that channel formation and translocation of the AC domain through cellular membranes may represent two parallel and unrelated, if not mutually exclusive, membrane activities of CyaA (Osickova et al, 1999). Complementation experiments with CyaA fragments, consisting out of different length of the repeat domain and are incapable in channel formation, and the mutant ACT 11490, which lack half of the repeat domain and forms channels with wild type properties, confirmed this results. A mixture of ACT 1-1490 and the fragments 10061706 or 1490-1681 restore the full calcium activated channel forming activity. It was further revealed that the regions 1490-1535 and 1628-1681 are essential for calcium activated ACT channel formation (Bauche et al, 2006; Basler et al, 2007). The membrane potential influences the channel-forming activity of ACT in a way that positive potential results in pores with a defined size and a high activity, whereas at negative potential the channels are not well defined, have a reduced channel-forming activity and a very short lifetime (Bauche et al, 2006; Basler et al, 2007). Channels inserted at positive potential, show for applied positive and negative voltages an MB-JASS 2007 – Session III – Properties of Channels Formed by Bacterial Porins and Toxins asymmetric current-voltage relationship. For positive potential, the current increased exponentially whereas for negative potential the current remains on a constant level. The results clearly indicate that the channels properties are dependent of the applied potential, but it seems that a membrane potential is not absolutely necessary for a correct channel formation. Furthermore the single-channel conductance of ACT channels is strongly affected by pH, whereas the ion selectivity remains unaffected. At pH 5 the single-channel conductance is between 3–5 pS and 60 pS at pH 9 in 1 M KCl. The voltage- and pH-effect are not disturbed by the use of mutants lacking the adenylate cyclase- or repeat-domain, indicating that both domains are not involved in voltage and pH sensing (Basler et al, 2007). 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