“RTX Toxins: ” – Session III

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
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).
MB-JASS 2007 – Session III – Properties of Channels Formed by Bacterial Porins and Toxins
Andersen, C. (2003) Channel-tunnels: outer membrane components of type I secretion systems and
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