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The Cholesterol-Dependent Cytolysin Family of Gram-Positive Bacterial Toxins

February 2010
Sub-cellular Biochemistry 51:551-77

February 2010
51:551-77

DOI:10.1007/978-90-481-8622-8_20

Source
PubMed

Authors:

Alejandro P Heuck

University of Massachusetts Amherst

The cholesterol-dependent cytolysins (CDCs) are a family of beta-barrel pore-forming toxins secreted by Gram-positive bacteria. These toxins are produced as water-soluble monomeric proteins that after binding to the target cell oligomerize on the membrane surface forming a ring-like pre-pore complex, and finally insert a large beta-barrel into the membrane (about 250 A in diameter). Formation of such a large transmembrane structure requires multiple and coordinated conformational changes. The presence of cholesterol in the target membrane is absolutely required for pore-formation, and therefore it was long thought that cholesterol was the cellular receptor for these toxins. However, not all the CDCs require cholesterol for binding. Intermedilysin, secreted by Streptoccocus intermedius only binds to membranes containing a protein receptor, but forms pores only if the membrane contains sufficient cholesterol. In contrast, perfringolysin O, secreted by Clostridium perfringens, only binds to membranes containing substantial amounts of cholesterol. The mechanisms by which cholesterol regulates the cytolytic activity of the CDCs are not understood at the molecular level. The C-terminus of perfringolysin O is involved in cholesterol recognition, and changes in the conformation of the loops located at the distal tip of this domain affect the toxin-membrane interactions. At the same time, the distribution of cholesterol in the membrane can modulate toxin binding. Recent studies support the concept that there is a dynamic interplay between the cholesterol-binding domain of the CDCs and the excess of cholesterol molecules in the target membrane.

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Chapter 20 Submitted July 2009

The original publication is available at:

https://link.springer.com/chapter/10.1007%2F978-90-481-8622-8_20

The cholesterol-dependent cytolysins family of Gram-positive bacterial

toxins

Alejandro P. Heuck, Paul C. Moe, and Benjamin B. Johnson

Department of Biochemistry and Molecular Biology, University of Massachusetts,

Amherst, MA 01003, U.S.A. heuck@biochem.umass.edu

Abstract

The cholesterol-dependent cytolysins (CDC) are a family of β-barrel pore-

forming toxins secreted by Gram-positive bacteria. These toxins are produced as water-

soluble monomeric proteins that after binding to the target cell oligomerize on the

membrane surface forming a ring-like pre-pore complex, and finally insert a large

β-barrel into the membrane (about 250 Å in diameter). Formation of such a large

transmembrane structure requires multiple and coordinated conformational changes. The

presence of cholesterol in the target membrane is absolutely required for pore-formation,

and therefore it was long thought that cholesterol was the cellular receptor for these

toxins. However, not all the CDC require cholesterol for binding. Intermedilysin, secreted

by Streptoccocus intermedius only binds to membranes containing a protein receptor, but

forms pores only if the membrane contains sufficient cholesterol. In contrast,

perfringolysin O, secreted by Clostridium perfringens, only binds to membranes

containing substantial amounts of cholesterol. The mechanisms by which cholesterol

regulates the cytolytic activity of the CDC are not understood at the molecular level. The

C-terminus of perfringolysin O is involved in cholesterol recognition, and changes in the

conformation of the loops located at the distal tip of this domain affect the

toxin-membrane interactions. At the same time, the distribution of cholesterol in the

membrane can modulate toxin binding. Recent studies support the concept that there is a

dynamic interplay between the cholesterol-binding domain of the CDC and the excess of

cholesterol molecules in the target membrane.

Keywords

Cholesterol, membranes, pore-forming toxins, cholesterol-dependent cytolysins,

membrane structure, cholesterol activity, transmembrane beta-barrel, transmembrane

pore, fluorescence spectroscopy, perfringolysin, lipid cluster.

Abbreviations

CDC, cholesterol-dependent cytolysins; PFO, perfringolysin O; ILY, intermedilysin;

PLY, pneumolysin; SLO, streptolysin O; anthrolysin; ALO; TMH/s, transmembrane β-

hairpin/s; D4, domain 4; L1, L2, and L3, loop 1, loop 2 and loop 3.

1. INTRODUCTION

The cholesterol-dependent cytolysins (CDC) are a growing group of β-barrel

pore-forming toxins secreted by Gram-positive bacteria (Farrand et al., 2008, Gelber et

al., 2008, Heuck et al., 2001, Jefferies et al., 2007, Mosser and Rest, 2006), and the first

members were discovered more than a century ago (see Alouf et al., 2006 for a historical

background on the CDCs). To date, there are complete amino acid sequences for 28

species distributed among the phyla of Firmicutes (genera of Bacillus, Paenibacillus,

Lysinibacillus, Listeria, Streptococcus, and Clostridium), and of Actinobacteria (genera

of Arcanobacterium and Gardenella) (Table 1). Most of the CDC have a cleavable signal

sequence and are therefore secreted to the extracellular medium via the general secretion

system (Sec, Harwood and Cranenburgh, 2008). A few exceptions are species of the

genus Streptoccocus (S. pneumoniae, S. mitis, and S. pseudoneumoniae) that produce

CDC without a signal sequence. The secretion mechanism for these CDC is unclear

(Jefferies et al., 2007, Marriott et al., 2008). After secretion to the extracellular medium,

the CDC fold into water-soluble monomeric proteins, travel and bind to the target

membrane, and oligomerize on the membrane surface forming characteristic arcs and

ring-like structures which are responsible for cytolysis. Several reviews have been

published describing the recent advances in the structural and mechanistic studies of the

CDC (Alouf et al., 2006, Giddings et al., 2006, Gilbert, 2005, Rossjohn et al., 2007,

Tweten, 2005). Here, we will focus on the role played by cholesterol during the

transformation of the CDC from a water-soluble monomer to a membrane-inserted

oligomeric complex. Although the cholesterol-dependent inhibition of the activity for

these toxins was one of the first biochemical properties attributed to the family

(Arrhenius, 1907), the molecular mechanism of the cholesterol-toxin interaction remains

as one of the least understood aspects in the study of the CDC toxin family.

TABLE 1

2. MECHANISM OF PORE-FORMATION

The 28 CDC family members listed in Table 1 show a significant degree of amino

acid identity (from 28.1 to 99.6 percent) and similarity (greater than 45.7 percent), with

amino acid sequences ranging from 471 to 665 amino acids in length. A comparison of

the primary structure of these proteins shows that they share a very low degree of

similarity at their N-terminus, in part because different species employ distinct signal

sequences for secretion, but also because some of the CDC members possess additional

domains located in this region (e.g., Farrand et al., 2008). If we consider just the

conserved core shared by all CDC and required for pore-formation activity [amino acids

38-500 in perfringolysin O (PFO)], the amino acid identity and similarity among different

members becomes higher than 36.7 and 58 percent, respectively (sequence length of

analyzed sequences range from 462 to 469, Figure 1). Therefore, from the analysis of the

primary structure of these toxins we can anticipate that all the CDC will exhibit similar

activities and three-dimensional structures.

FIGURE 1

The first crystal structure for a CDC was solved for PFO by Rossjohn and

colleagues (1997). The crystal structure for two other CDC, intermedylisin (ILY) and

anthrolysin (ALO), have been solved so far, and all of them share similar secondary and

tertiary structure (Bourdeau et al., 2009, Polekhina et al., 2005). They have a high

β-strand content and their structures have been divided into four domains, with the

C-terminal domain (domain 4 or D4) being the only independent and continuous domain

(Figure 2A) (Polekhina et al., 2006).

PFO secreted by pathogen Clostridium perfringens is a prototypical CDC (Tweten

et al., 2001). To describe the general mechanism of pore-formation for the CDC we will

depict the current knowledge of the PFO cytolytic mechanism which starts with the

binding of the toxin to the target membrane, and concludes with the insertion of a large

transmembrane β-barrel (Figure 2A).

FIGURE 2

Upon encountering a cholesterol-containing membrane, PFO oligomerizes and

spontaneously inserts into the bilayer to form a large transmembrane pore (~35-50

monomers per oligomer; approximately 250 Å in diameter, Figure 2), (Czajkowsky et al.,

2004, Dang et al., 2005, Mitsui et al., 1979, Olofsson et al., 1993). The C-terminus of

PFO (D4) encounters the membrane first (Figure 2A, I, Heuck et al., 2000, Nakamura et

al., 1995, Ramachandran et al., 2002). The binding of D4 triggers the structural

rearrangements required to initiate the oligomerization of PFO monomers

(Ramachandran et al., 2004, Soltani et al., 2007a) and formation of a pre-pore complex

on the membrane surface (Figure 2A, II, Heuck et al., 2003, Shepard et al., 2000, Tilley

et al., 2005). Pore formation commences when two amphipathic β-hairpins from each

PFO molecule insert and span the membrane (Figure 2A, III, Hotze et al., 2002,

Shatursky et al., 1999, Shepard et al., 1998). The concerted insertion of two

transmembrane β-hairpins (TMHs) from ~35 PFO monomers then creates a large

transmembrane β-barrel that perforate the membrane (Dang et al., 2005, Tilley et al.,

2005). This general mechanism of pore-formation is followed by most CDC, however,

some variations have been observed for specific members and they will be described in

the following sections.

2.1 Localizing the target membrane

The first step in the CDC cytolytic cascade is the recognition of the target cell

(Figure 2A, I). The CDC bind to the target membrane by recognizing a specific

membrane lipid, cholesterol, or by recognizing a membrane-anchored protein as in the

case of ILY (Giddings et al., 2004). Cholesterol-recognition provides specificity towards

eukaryotic cells in general, and the glycosylphosphatidylinositol-anchored protein CD59

provide specificity for human cells. While it has been shown that ILY interacts with the

CD59 receptor forming a 1:1 complex (Lachapelle et al., 2009), the interaction of other

CDC with cholesterol is less understood. Independently of the recognition mechanism, it

appears that all CDC bind to the target membranes via D4 (Nagamune et al., 2004,

Soltani et al., 2007a).

2.2 Grouping forces on the membrane surface: pre-pore formation

After successful recognition of the target membrane, the CDC oligomerize in the

membrane surface to form a membrane-bound pre-pore complex (Figure 2, II).

Formation of a pre-pore complex seems to be a common feature of the β-barrel pore-

forming toxins (Heuck et al., 2001, Miller et al., 1999, Shepard et al., 2000, Walker et al.,

1992). The secreted monomeric proteins do not oligomerize in solution, and it has been

shown that the binding of the toxins to the target membrane is required to trigger the

monomer-monomer association (Abdel Ghani et al., 1999, Lachapelle et al., 2009,

Ramachandran et al., 2004). Although oligomerization has been observed in the absence

of membranes for certain CDC (e.g., pneumolysin, (PLY) Gilbert et al., 1998, Solovyova

et al., 2004), it only occurs when the toxin concentration is relatively high (in the

micromolar range or higher) compared to the concentration needed for efficient

oligomerization when incubated with natural membranes. The difference in efficiency

between oligomerization in solution and at the surface of a cell membrane suggests that

the cells in some way promote the association of toxin monomers. In general,

oligomerization of β-barrel pore-forming toxins requires the exposure of hidden

polypeptide regions involved in the monomer-monomer interaction (Heuck and Johnson,

2005, Heuck et al., 2001). In the CDC, this process is triggered by conformational

changes induced by protein-lipid interactions (e.g., PFO, Ramachandran et al., 2004) or

by conformational changes induced by protein-protein interactions (e.g., ILY Lachapelle

et al., 2009).

Ramachandran et al. (2004) have shown that in the water-soluble form of the

toxin, oligomerization is prevented by blocking access to one edge of a core β-sheet in

the monomer (Figure 2B). This blockage prevents its association with the edge of the

core β-sheet in the neighboring monomer, thus impeding formation of an extended

β-sheet. Specifically, premature association of PFO molecules (before they bind to the

appropriate membrane surface) is prevented by the presence of β5, a short polypeptide

loop that hydrogen bonds to β4 in the monomer, and thereby prevents its interaction with

the β1 strand in the adjacent monomer. This feature is conserved in all crystal structures

reported for the CDC (i.e., PFO, ILY, and ALO).

The structural changes associated with converting a CDC from a water-soluble

monomer to a membrane-inserted oligomer extend through much of the molecule. The

binding of D4 to the membrane surface immediately elicits a conformational change in

domain 3, more than 70 Å above the membrane (Abdel Ghani et al., 1999, Heuck et al.,

2000, Ramachandran et al., 2002, Ramachandran et al., 2004, Ramachandran et al.,

2005). This conformational change rotates β5 away from β4 and thereby exposes β4 to

the aqueous medium where it can associate with the always-exposed β1 strand of another

PFO molecule to initiate and promote oligomerization (Figure 2B).

Such an extensive network of structural linkages within a CDC can be

advantageous because it reduces the chance of prematurely entering a structural transition

that exposes a TMH. By allosterically linking different domains or regions of the protein,

the system can couple separate interactions (e.g., binding to the membrane and binding to

another subunit) and thereby ensure that pore formation proceeds only when the

necessary criteria are met. Given the important allosteric communication between the

membrane binding domain and the pore-forming domain, it is not surprising that the most

conserved regions on these proteins are located among inter-domain segments, forming

an almost continuous path with its origin at the tip of D4 and terminus at the segments

that form the amphipathic TMHs (Figure 3). Interestingly, while most of the surface

exposed residues of the CDC are not very conserved, the residues at the surface of the D4

tip, involve in membrane interaction, are highly conserved.

FIGURE 3

Establishment of an oligomeric complex in the membrane surface facilitates the

formation of a transmembrane pore because the insertion of a single amphipathic

β-hairpin into a membrane is not energetically favored. In a hydrophobic environment

that lacks hydrogen bond donors or acceptors, isolated β-hairpins cannot achieve the

hydrogen-bond formation necessary to lower the thermodynamic cost of transferring the

polar atoms of the polypeptide backbone into the hydrocarbon interior (White and

Wimley, 1999). However, this energy barrier is circumvented if the β-strands are inserted

as β-sheets and form closed structures such as a β-barrel. For monomeric β-barrel

membrane proteins such as OmpA, a concerted folding mechanism has been observed in

vitro in which the hydrogen bonds formed between adjacent β-chains presumably favor

the insertion of the β-barrel into the membrane (Kleinschmidt, 2006, Tamm et al., 2004).

Similarly, the formation of a pre-pore complex may be required to allow the concerted,

and perhaps simultaneous, insertion of the β-hairpins from individual monomers, thereby

overcoming the energetic barrier of inserting non-hydrogen-bonded β-strands into the

membrane. Whereas it is clear that the formation of a complete ring (or pre-pore

complex) on the membrane surface will minimize the energetic requirements for inserting

a β-barrel into the membrane, it is likely that the insertion of incomplete rings can also

occur if monomer recruitment into the oligomer slows down. In the absence of additional

monomers, the incomplete pre-pore complexes observed in vitro (or metastable arc

structures) will be trapped, and they may have enough time to insert into the membrane

and form a pore (Gilbert, 2005). Insertion of an arc may well form a transmembrane pore

by itself, or in association with other arcs (double arc structures, Palmer et al., 1998). A

minimal number of monomers must be required to overcome the energetic barrier of

inserting an arc-like β-sheet into the membrane. It has been shown that independently of

the toxin/lipid ratio, the pores formed by PFO and streptolysin O (SLO) are at least large

enough to allow the passage of proteins with an approximate diameter of 100 Ǻ (Heuck

et al., 2003).

In summary, a coordinated train of events regulates the proper assembly of the

CDC oligomeric complex at the surface of the target membrane. Formation of these

oligomeric structures facilitates the insertion of numerous TMHs which are required to

form the large transmembrane β-barrel.

2.3 Perforating the membrane: insertion of a large β-barrel

A characteristic of the CDC that distinguishes them from most other β-barrel

pore-forming toxins is the use of two amphipathic β-hairpins per monomer to form the

large transmembrane barrel (Heuck and Johnson, 2005, Heuck et al., 2001, Shatursky et

al., 1999). In the water-soluble monomeric configuration of the CDC these TMHs are

folded as short α-helices presumably to minimize the exposure of the hydrophobic

surfaces (Heuck and Johnson, 2005). These helices, located at either side of the central

β-sheet in domain 3, extend and insert into the membrane bilayer (Shatursky et al., 1999,

Shepard et al., 1998). The conversion of short α-helices to amphipathic β-hairpins

constituted a new paradigm for how pore-forming toxins transform from a water-soluble

to membrane-inserted conformation. This structural transformation has been recently

found in eukaryotic pore-forming proteins, as revealed by the structure of the membrane

attack complex/perforin superfamily members (Hadders et al., 2007, Rosado et al., 2007).

After insertion the hydrophobic surfaces of the TMHs are exposed to the non-polar lipid

core of the membrane and the hydrophilic surfaces face the aqueous pore. A concerted

mechanism of insertion ensures that the hydrophilic surfaces of the hairpins remain

exposed to the aqueous medium, and not to the hydrophobic core of the membrane. Such

a coordinated insertion requires the displacement of lipids as the aqueous pore is formed

in the membrane.

The creation of a circular hole, having a radius of nearly 150 Å, in a liposomal

membrane requires the displacement of about 1000 phospholipid molecules in each

leaflet (or about 800 phospholipids plus 800 cholesterol molecules because the average

surface area occupied by one phospholipid molecule plus one cholesterol molecule is ~90

Å2 in a 1:1 phospholipid/cholesterol mixture (Heuck et al., 2001, Lecuyer and

Dervichian, 1969)). Analysis of the release of markers encapsulated in liposomes when

using limiting concentrations of PFO or SLO showed that both, the small markers and the

large markers are released with the same rate. Therefore, it appears that all of these lipid

molecules leave the pore formed by these CDC at the same time (Heuck et al., 2003),

though not all agree (Palmer et al., 1998).

A direct comparison of the cytolytic mechanism of PFO and ILY showed that

whereas ILY does not require cholesterol for binding, pore-formation is entirely

dependent on the presence of cholesterol in the target membrane (Giddings et al., 2003).

Employing a series of ILY mutants that block pore formation at different stages, Hotze

and colleagues have shown that ILY remains engaged with its receptor (human CD59)

throughout the assembly of the pre-pore complex, but it is released from CD59 upon the

transition to the membrane-inserted oligomer (Lachapelle et al., 2009). Upon release

from the receptor, ILY is anchored to the membrane via D4 suggesting that this domain

still conserves the cholesterol binding properties of other CDC members (note that

insertion of the ILY β-barrel does not occur if cholesterol is depleted from the

membrane).

After pre-pore formation, the insertion of the PFO TMHs requires the proper

intermonomer β-strand alignment. Ramachandran et al. (2004) suggested that the

π-stacking interaction between Y181 and F318 guides the alignment of the TMHs of

adjacent monomers (Figure 2B). Interestingly, while Y181 is completely conserved in the

28 members of the CDC family, F318 is not. Instead of phenylalanine, this position is

occupied by valine in lectinolysin, vaginolysin, and PLY, by isoleucine in ILY, and

alanine in pyolysin. It will be interesting to determine if a mutation of the conserved

PFO-Y181-equivalent in ILY results in a pre-pore blocked derivative, as observed in

PFO.

3. THE ROLE OF CHOLESTEROL IN MEMBRANE BINDING

Among all the different lipids that shape the vast diversity of cell membranes,

cholesterol is a distinguishing feature of mammalian cells. The CDC have evolved to take

advantage of this feature of mammalian membranes, and their ability to perforate the

target membrane is totally dependent on the presence of cholesterol (Giddings et al.,

2003, Palmer, 2004).

In liposomal membranes containing only phosphatidylcholine and cholesterol,

more than 30 mole % cholesterol is required for CDC such as tetanolysin (Alving et al.,

1979), SLO (Rosenqvist et al., 1980), and PFO (Heuck et al., 2000, Ohno-Iwashita et al.,

1992), to bind and create a pore in the bilayer. For PFO, no binding at all is detected

when the cholesterol concentration in the liposomal membrane is less than ~30 mole % of

the total lipids (Flanagan et al., 2009, Heuck et al., 2000, Nelson et al., 2008). Thus, if

cholesterol acts solely as a receptor, and hence as a PFO binding ligand, reducing the

cholesterol concentration in the bilayer should only affect the kinetics of the cytolytic

process. In other words, lowering the amount of cholesterol in the membrane should

result in a longer time required for PFO to form a transmembrane pore. However, the

sharp transition observed in the binding isotherm of PFO suggests that the basis of this

recognition is more complex than a simple encounter frequency between PFO and

individual cholesterol molecules (Heuck et al., 2000).

3.1 Domain 4 and membrane recognition

The initial members of the CDC family were characterized by their sensitivity to

oxygen and cholesterol (Alouf et al., 2006). Toxins isolated from culture supernatants

were inactivated by exposure to oxygen present in the air or when pre-incubated with

cholesterol. While the oxygen-dependent inactivation of the toxins could be reversed by

incubation with thiol-based reducing agents, inactivation by pre-incubation with

cholesterol was not reversible. A direct consequence of these findings was that the

discovery of new CDC members was strongly influenced by the search for these two

distinguishing features in the newly encountered hemolytic toxins: inhibition by oxygen

and cholesterol. Therefore, it is not surprising that the first sequences obtained for the

CDC revealed that all of them contained a conserved undecapeptide which was critical

for cholesterol recognition, and a unique cysteine in this segment that was sensitive to

aerobic oxidation. This correlation led researchers to postulate that the conserved

undecapeptide, and attendant cysteine constituted the cholesterol binding site for the

CDC. However, advancements in recombinant DNA technology soon allowed

researchers to show that this unique cysteine was not essential for cholesterol recognition.

First, the replacement of this cysteine with alanine rendered a protein that remained

hemolytic (Michel et al., 1990, Pinkney et al., 1989, Saunders et al., 1989, Shepard et al.,

1998). Second, the sequence of newly discovered CDC members showed that this

cysteine was indeed replaced by alanine during the evolution of different Gram-positive

species (Billington et al., 2001, Nagamune et al., 2000).

New protein homologues of the CDC are being revealed as new genomes are

sequenced, and these new family members are showing more variability in the amino

acid sequence of this segment. The multi-sequence alignment for the 28 CDC sequences

shows that 20% of the CDC contain amino acid substitutions in the undecapeptide. Based

on this newly accumulated evidence, the original view of the conserved undecapeptide as

the cholesterol binding site is being replaced by alternative models for membrane-

binding. It has been shown that one of the CDC, intermedilysin (ILY) recognize the

target membrane by the specific binding to a human protein receptor, CD59, and it is

therefore possible that other members may also bind to the target membrane by as yet

unidentified protein receptors (Bourdeau et al., 2009). In addition to the undecapeptide,

other well conserved peptide loops located at the tip of D4 may contribute to the

cholesterol recognition motif (Ramachandran et al., 2002, Soltani et al., 2007a, Soltani et

al., 2007b).

3.1.1 The conserved loops

PFO D4 has a 4 stranded β-sandwich structure that interacts with the membrane

surface only at one end via the distal loops that interconnect the eight β-strands that form

the domain (Figure 4A Ramachandran et al., 2002, Rossjohn et al., 1997, Soltani et al.,

2007a). Superimposition of the D4 α-carbons for PFO, ALO, and ILY reveals that the

global structure of D4 is well conserved among these members. The main differences

arise in the conformation of the undecapeptide, involved in toxin-membrane interaction,

and in the loops that are close to the domain 2-D4 interface (Figure 4A).

FIGURE 4

Three of the four loops located at the distal tip of D4 are highly conserved among

the CDC members: the conserved undecapeptide (also known as the Trp-rich loop), L1,

and L2 (Figure 4B). The L3 loop is less conserved and is located farther away from the

unique cysteine residue. Recent data obtained by Tweten and colleagues suggest that in

addition to the undecapeptide, the other D4 loops (L1-L3) may also play a role in the

cholesterol-dependent recognition of the CDC (Soltani et al., 2007b). Single amino acid

modifications in these loops prevented the binding of PFO to cholesterol-rich liposomes,

and abolished the pre-pore to pore transition for ILY in a cholesterol-dependent manner.

Both of these events involve the association of the D4 with the cholesterol-containing

membrane. It has become clear that the three-dimensional arrangement of the

undecapeptide and the L1-L3 loops is important for the association of the CDC with the

cholesterol-containing membrane (Giddings et al., 2003, Polekhina et al., 2005, Soltani et

al., 2007a, Soltani et al., 2007b).

Interestingly, changes in the pH of the medium which affect the conformation of

D4 also influence the cholesterol-toxin interaction. A reduction of the pH from 7.5 to 6.0

induces a conformational change in PFO causing the tryptophan residues to be more

exposed to the aqueous solvent, and also alters the threshold for the minimal cholesterol

concentration required to trigger binding of PFO to liposomal membranes (Nelson et al.,

2008). Since no major changes are expected to occur in the structure of the membrane in

between pH 7.5 and 6.0, one can assume that protonation of certain amino acids in PFO

may alter the D4 conformation, and as a consequence, its ability to recognize cholesterol

in the target membrane. A related effect has been observed for listeriolysin O (LLO), a

CDC recognized for having an optimum acidic pH for activity (Bavdek et al., 2007). The

loss of activity for LLO at neutral pH can be rescued by increasing the concentration of

cholesterol in the membrane.

Given that conformational changes in D4 can alter the cholesterol-dependent

properties of the CDC, one can speculate that the conformational change triggered by the

binding of ILY to the CD59 receptor (Soltani et al., 2007a), may modulate the

cholesterol-dependent association with the membrane required for pore-formation.

Unfortunately, despite the various high-resolution structures available for the

CDC, and the multiple functional data obtained by modification of amino acids located at

the D4 loops, it is still unclear how cholesterol modulates the conformational changes

required to anchor the toxin to the membrane and to insert a large transmembrane

β-barrel. Furthermore, is no clear if the binding of PFO (and related CDC) is triggered by

the binding of a single cholesterol molecule (Geoffroy and Alouf, 1983, Nollmann et al.,

2004, Polekhina et al., 2005), or by the recognition of a more complex cholesterol-

arrangement in the bilayer structure (Bavdek et al., 2007, Flanagan et al., 2009, Heuck

and Johnson, 2005, Heuck et al., 2007, Nelson et al., 2008).

3.2 Searching for cholesterol in the membrane

The binding of a protein domain to a membrane surface is in general, a two step

process that involves the initial formation of non-specific collisional complex followed

by the formation of a tightly bound complex. The first step is diffusional and may involve

electrostatic interactions, and the second step stabilizes the initial interaction by

membrane penetration of non-polar amino acids and/or specific interactions between the

protein and the membrane lipids (Cho and Stahelin, 2005). The initial membrane

association locates non-polar amino acids close to the interfacial region of the bilayer,

facilitating their exposure to the hydrophobic core. Non-polar amino acids are not usually

exposed to the protein surface, and therefore conformational changes are required to

expose them to the membrane.

Exposure of the aromatic residues located in the undecapeptide occurs upon

membrane binding, though they do not penetrate deeply into the bilayer core (Heuck et

al., 2003, Nakamura et al., 1998, Sekino-Suzuki et al., 1996). The sensitivity of the

undecapeptide to amino acid changes suggests that the exposure of aromatic amino acids

and membrane binding requires precise conformational changes and/or a particular three-

dimensional conformation. A conformational change in the undecapeptide that modulate

cholesterol binding and membrane anchoring has been suggested for PFO (Rossjohn et

al., 1997), however the binding site for cholesterol, if any, remains elusive.

It has become apparent that in addition to the three dimensional structure of the

binding-domain, the arrangement of the cholesterol molecules in the bilayer is also

critical for successful binding. In a membrane, the cholesterol molecule swims in the

non-polar core of the bilayer with an orientation nearly parallel to the acyl chains of the

phospholipids. The non-polar hydrocarbon tail of the molecule orients towards the center

of the bilayer, and the 3-β-OH group locates close to the ester bonds formed by the fatty

acid chains and the glycerol backbone of the phospholipids near the membrane-water

interface. Compared to the phospholipid head groups, the polar group of the cholesterol

molecule is not very exposed to the membrane surface. Therefore, it is not strange that at

relatively low concentrations the cholesterol molecules are not readily available to

interact with water-soluble molecules (e.g., cholesterol oxidase, cyclodextrins) (Lange et

al., 1980).

3.2.1 Cholesterol availability in membrane bilayers

In multicomponent membranes, the availability of cholesterol at the membrane

surface is regulated by the interactions between cholesterol and other the components of

the membrane (phospholipids, glycolipids, proteins). The more the cholesterol interacts

with the membrane components, the less available it will be to interact with extra-

membranous molecules. Factors that affect the interaction of cholesterol with

phospholipids are the length of the acyl chains, the presence of double bonds in these

chains, the size of the polar head-groups, and the ability of the phospholipid to form

hydrogen bonds with the hydroxyl group of cholesterol (Ohvo-Rekilä et al., 2002).

When cholesterol is added to a membrane containing a single phospholipid

species, two phases appear in a concentration-dependent manner (Mouritsen and

Zuckermann, 2004, Sankaram and Thompson, 1991). This suggests that instead of

randomly distributing among the membrane phospholipids, cholesterol associates with

the phospholipids presumably forming stoichiometric complexes (Radhakrishnan and

Mcconnell, 1999). When the phospholipids are in excess, most of the cholesterol

molecules are forming complexes with phospholipids. These complexes are immiscible

in the pure phospholipid phase and therefore a two-phase mixture appears in the

membrane. Increasing the cholesterol concentration will increase the population of the

complexes until they form a single phase containing the complexes with a minor presence

of uncomplexed phospholipids and cholesterol molecules. Beyond this point, the added

cholesterol molecules (free cholesterol) will mix with the complexes until they reach the

solubility limit and precipitate out of the membrane (Mason et al., 2003). Cholesterol

molecules do not form stable bilayers in aqueous solution, so when present in excess they

cannot form a new stable and extended phase. The free cholesterol molecules in excess

have a tendency to “fly” away from the membrane, and outside the membrane they will

be prone to aggregate and form crystals (Harris, 1988).

The formation of phospholipid-cholesterol complexes can explain the low

interaction detected between cyclodextrins and cholesterol when the sterol is present in

low amounts (Mcconnell and Radhakrishnan, 2003). An alternative model to account for

this behavior was proposed by Huang and Feigenson (1999). These authors propose that

the hydrophobic effect positions the phospholipid head groups toward the membrane

surface to protect the hydrophobic molecule of cholesterol from the unfavorable contact

with water. When the concentration of cholesterol in the membrane achieves and exceeds

the protective capacity of the head-groups, the tendency for the sterol molecules to “fly”

away will increase.

Both models provide a reasonable explanation for the increased accessibility of

cholesterol at high sterol/phospholipid ratios, and the consensus is that they are not

mutually exclusive (Lange and Steck, 2008, Mesmin and Maxfield, 2009). Binding

(and/or pore-formation) of the CDC occurs at high cholesterol concentration where free

cholesterol become available, and therefore any of these models can be used to explain

the experimental observations.

In more complex lipids mixtures, when more than one phospholipid is present in

the membrane, the total cholesterol content will distribute unevenly between any formed

phases (Goñi et al., 2008, Veatch and Keller, 2002). How much cholesterol is present in

each phase will be governed by the interaction between cholesterol and the components

(lipids and proteins) present in the phases (Epand, 2006).

3.2.2 The role of other lipids

The pioneering work of Ohno-Iwashita and colleagues in the binding of PFO to

membranes showed that the phospholipid composition affects the arrangement of

cholesterol in the membrane. Using a protease-nicked derivate of PFO they showed that

the binding of the toxin was no only influenced by the total amount of cholesterol present

in the membrane, but also by the phospholipid composition. They found that this PFO

derivative preferentially binds to cholesterol-rich membranes composed of phospholipids

with 18-carbon acyl chains (Ohno-Iwashita et al., 1992, Ohno-Iwashita et al., 1991). An

effect on cholesterol state in the membrane by ceramides and glycerolipids was also

suggested by Zitzer et al. (2003) based on their studies of SLO pore-formation in

liposomal membranes prepared with different phospholipids. Lipids having a conical

molecular shape appear to effect a change in the energetic state of membrane cholesterol

that in turn augments the interaction of the sterol with the cholesterol-specific cytolysin.

Interestingly, these authors also showed that SLO was active when membranes were

prepared solely with the enantiomeric cholesterol, suggesting that the effect associated

with the presence of cholesterol may be other than a site specific binding event (Zitzer et

al., 2003).

A more systematic analysis of the interaction of PFO D4 with membranes

prepared with different phospholipds and sterols revealed that PFO binding to the bilayer

and the initiation of the sequence of events that culminate in the formation of a

transmembrane pore depend on the availability of free cholesterol at the membrane

surface (Flanagan et al., 2002, Flanagan et al., 2009, Nelson et al., 2008). These studies

also showed that changes in the acyl chain packing of the phospholipids, and cholesterol

in the membrane core do not correlate with PFO binding. Taken together, all these studies

suggest than the binding of PFO (and SLO) to the membrane is triggered when the

concentration of cholesterol exceeds the association capacity of the phospholipids, and

this cholesterol excess is then free to associate with the toxin (Figure 5).

FIGURE 5

The requirement of such high cholesterol content in membranes was initially

associated with the binding of PFO to cholesterol-rich domains (or membrane rafts)

(Ohno-Iwashita et al., 2004, Waheed et al., 2001). However, recent results indicate that

this assertion may require further analysis and consideration. It was found that the

incorporation of sphingomyelin, a necessary component for the formation of membrane

rafts, inhibited rather than promoted the binding of PFO to membranes (Flanagan et al.,

2009). No correlation was found between PFO binding, and the amount of the detergent-

resistant fraction in membranes, a fraction usually associated with membrane rafts

(Flanagan et al., 2009). Incorporation of sterols that promote the formation of ordered

membrane domains was not critical to promoting the PFO-membrane interaction (Nelson

et al., 2008). Therefore, one needs to be cautious when employing PFO as a probe to

reveal the presence of membrane rafts in cellular membranes. Rather than recognizing a

particular membrane “raft”, PFO seems to bind to membranes containing free cholesterol

(or where cholesterol has a high chemical activity).

3.2.3 Cholesterol is enough

It was long known that incubation of SLO (Duncan and Schlegel, 1975, Johnson

et al., 1980), PFO (Mitsui et al., 1979), cereolysin (Cowell and Bernheimer, 1978),

alveolysin (Johnson et al., 1980), PLY (Johnson et al., 1980), and LLO (Vazquez-Boland

et al., 1989) with cholesterol dispersed in aqueous solution produced the typical

aggregated sterol-toxin complexes. For PFO and SLO, typical ring- and arc-like

structures were observed after incubation with cholesterol at concentrations above its

solubility limit (i.e., higher than 5 μM Duncan and Schlegel, 1975, Haberland and

Reynolds, 1973, Harris et al., 1998, Mitsui et al., 1979).

To clarify the role of cholesterol in PFO cytolysis, the extent to which the

different steps of the cytolytic mechanism could be elicited solely by the presence of

cholesterol was analyzed (Heuck et al., 2007). Using site-directed fluorescence labeling

of PFO in combination with multiple independent fluorescence techniques (Heuck and

Johnson, 2002, Johnson, 2005), it was revealed that a selective interaction between the

undecapeptide and the D4 loops with cholesterol dispersed in aqueous solution is

indistinguishable from the interaction of PFO with cholesterol-containing membranes.

Binding solely to cholesterol aggregates in aqueous solution is sufficient to initiate the

coupled conformational changes that extend throughout the toxin molecule from the tip

of D4 to the TMHs. Moreover, it was found that the topology of D4 bound to cholesterol

aggregates was identical to the one observed in liposomal membranes, and that the

binding of PFO to cholesterol aggregates was sufficient to trigger the conformational

change in domain 3 that has been associated with oligomerization (Heuck et al., 2007,

Ramachandran et al., 2004). As previously observed for SLO in cholesterol micro-

crystals (Harris et al., 1998), oligomerization and formation of typical arc and ring

structures were observed in the presence of cholesterol aggregates. Surprisingly, none of

these changes were produced by epicholesterol, a sterol that differs from cholesterol only

in that the hydroxyl group is directed axially instead of equatorial (Heuck et al., 2007).

Taking advantage of the inability of PFO to recognize epicholesterol, competition

experiments were done to examine how cholesterol packing in the bilayer affects the

interactions with the membrane. More than 48 mole% cholesterol is required for PFO to

bind to POPC-cholesterol liposomes (Flanagan et al., 2009). However, when the

epicholesterol was mixed with cholesterol to maintain the concentration of total sterols

constant at 48 mole%, and to reduce the net amount of cholesterol in the membrane, it

was shown that in this case considerable binding of PFO was found with as little as 19

mole% cholesterol. Epicholesterol apparently intercalates in the bilayer and competes

with cholesterol for association with phospholipids, as reported for other membrane

intercalating agents (Lange et al., 2005). These data therefore confirmed that there are at

least two distinctive states of cholesterol in a typical membrane bilayer: one in which

cholesterol is readily accessible for binding to proteins such as PFO (free cholesterol),

and one in which the sterol is associated with surrounding membrane components that

reduce its exposure to the surface (e.g., phospholipid headgroups may obscure access to

sterols associated with phospholipid acyl chains).

The selective binding of PFO to cholesterol aggregates and not to epicholesterol

aggregates, suggests that the failure to bind epicholesterol when incorporated in

membrane bilayers is not related to the packing or association of this sterol with the

phospholipids. This failure is rather caused by the “wrong” orientation of the hydroxyl

group (Murari et al., 1986), which it may be required for the specific docking of the sterol

molecule to a binding pocket located in D4 (Figure 5B Rossjohn et al., 2007). or

alternatively, to be properly exposed at the surface of a lipid cluster that may act as a

platform for the anchoring of the D4 loops (Figure 5C). Such a cluster may be preformed

on the membrane before binding, or formed as a result of the interaction of D4 with the

bilayer surface. Redistribution of lipids after protein-binding has been observed for LLO

(Gekara et al., 2005), and other proteins (e.g., Heimburg et al., 1999).

The PFO and SLO specific binding to cholesterol aggregates and microcrystals

(Harris et al., 1998, Heuck et al., 2007), together with the need for more than 30 mole%

cholesterol in membranes to trigger binding (Flanagan et al., 2009, Heuck et al., 2000,

Nelson et al., 2008), suggest that the role of cholesterol in the cytolytic mechanism of the

CDC may be more complex than solely binding to a specific binding site. An alternative

explanation would be the need of a cluster of cholesterol molecules at the membrane

surface to provide a docking platform for the D4 loops (Gekara et al., 2005, Heimburg et

al., 1999, Heuck and Johnson, 2005). Interestingly, the binding of pore-forming toxins to

lipid clusters have been reported for Staphylococcus aureus α-hemolysin (Valeva et al.,

2006), and the need for small cholesterol clusters have been recently suggested for the

binding of LLO to membranes (Bavdek et al., 2007). Further work is needed to

unambiguously determine the mechanism by which cholesterol specifically anchors the

CDC to the target membrane.

4. CONCLUSIONS AND FUTURE PERSPECTIVES

Recent studies support the concept that there is a complex interplay between the

structural arrangement of the D4 loops and the distribution of cholesterol in the target

membrane (Bavdek et al., 2007, Flanagan et al., 2009, Giddings et al., 2003, Heuck and

Johnson, 2005, Nelson et al., 2008, Polekhina et al., 2005, Ramachandran et al., 2002,

Soltani et al., 2007a, Soltani et al., 2007b). Modifications in the lipid composition alter

the cholesterol arrangement in the membrane, and as a consequence, the binding of the

CDC (Flanagan et al., 2009, Nelson et al., 2008). At the same time, modifications to the

structure of the CDC effected by mutations, changes in the pH of the medium, or other

factors, modifies the threshold for the amount of cholesterol required to trigger binding

(Bavdek et al., 2007, Nelson et al., 2008, Moe & Heuck, unpublished).

The presence of free cholesterol molecules at the membrane surface seems to be

critical to trigger the binding of most CDC. A direct inference from these findings is that

the exposure of cholesterol at the membrane surface may be facilitated by the action of

other membrane-damaging toxins secreted by these pathogens like, for example,

phospholipases C. These toxins cleave the head-groups of phospholipids, and

consequently increase the exposure of cholesterol molecules (or availability of free

cholesterol) to the membrane surface. Cooperation between the CDC and different

phospholipases C contribute to the pathogenesis of at least two organisms. A synergic

effect has been reported for the action of PFO and α-toxin in clostridial myonecrosis

(Awad et al., 2001), and both phospholipases C and LLO have been identified as key

factors for the vacuolar dissolution and cell-to-cell spreading mechanism of Listeria

monocytogenes (Alberti-Segui et al., 2007).

Complete understanding of the mechanism of pore formation for the CDC at the

molecular level would require high-resolution structures of the initial (water-soluble

monomer), the final (membrane-inserted oligomer), and any intermediate state involved

in the cytolytic process (including complexes with receptors or lipids). Great progress has

been achieved to this end, but there is much more to be accomplished. A few crystal

structures for monomeric CDC are currently available (PFO, ILY, ALO, Bourdeau et al.,

2009 , Polekhina et al., 2005, Rossjohn et al., 1997), and the low resolution structure for

the pre-pore complex and the membrane-inserted oligomer of PLY have been obtained by

cryo-electron microscopy (Tilley et al., 2005).

It has become clear that the analysis of complex biological systems, in particular

those involving membranes, benefits from the combination of high-resolution structural

techniques (e.g., X-ray crystallography, nuclear magnetic resonance, electron

microscopy) and spectroscopic analysis of probes incorporated at specific positions in the

proteins (e.g., electron paramagnetic resonance, fluorescence spectroscopy) (Cowieson et

al., 2008, Heuck and Johnson, 2002, Hubbell et al., 2000). In addition to providing

structural information, by monitoring the spectral signal of these probes as a function of

time, one can determine the kinetics of the discrete steps of the pore-formation

mechanism (Heuck et al., 2000, Heuck et al., 2003) and the dynamics of the structural

transformations (Columbus and Hubbell, 2002).

Understanding the CDC function in the establishment of the diseases caused by

various Gram-positive pathogens is far from complete (Marriott et al., 2008, Schnupf and

Portnoy, 2007). The actual role of the CDC in bacterial pathogenesis may be more

complex than merely forming a transmembrane pore. For example, it has been proposed

that SLO is involved in protein translocation during Streptococcus pyogenes infection

(Madden et al., 2001, Meehl and Caparon, 2004).

The involvement of protein receptors in the mechanism of certain CDC is another

area that require further investigation. The discovery of the ILY receptor illuminated two

distinct roles for cholesterol in the cytolytic mechanism of the CDC (Giddings et al.,

2003). ALO’s strong preference for targeting the apical side of gut epithelial cells

suggests that a receptor (other than cholesterol) may be present in these cells (Bourdeau

et al., 2009). Clearly, there is yet much to learn about the complex and fascinating roles

played by the CDC in bacterial pathogenesis.

ACKNOWLEDGMENTS

Work in the authors’ laboratory was supported by a Scientist Development Grant

from the American Heart Association to A.P.H.

Table 1. Homologs in Gram-positive species compose the CDC family. Twenty-eight

CDC family members from divergent phyla have been identified by amino acid sequence.

The protein three letter code for each homolog (as defined in figure 1) is followed by its

phylogenetic relationship to the PFO standard. Because many of the CDC family are

expressed with variable N-terminus, PFO relationship is expressed in bold for the

conserved core only (corresponding to amino acids 38-500 of PFO) and in parentheses

for the full length form. The lengths of the respective polypeptides are presented.

Percentages of identity and similarity were calculated as indicated in Figure 1 legend.

* subsp. equisimilis

PHYLUM

Firmicutes

CLASS

Bacilli

ORDER

Bacillales

FAMILY

Bacillaceae

GENUS

Bacillus %Identity %Similarity Length ID

SPECIES

B. anthracis ALO 72 (68) 88 (83) 462 (512) ZP_03017964.1

B. thurigiensis TLO 74 (69) 88 (83) 462 (512) YP_037419

B. cereus CLO 74 (69) 88 (84) 462 (512) YP_002369889.1

B. weihenstephanensis WLO 74 (69) 87 (83) 462 (512) ABY46062

Listeriaceae

Listeria

L. monocytogenes LLO 43 (40) 66 (62) 469 (529) ABH07645

L. seeligeri LSO 45 (41) 67 (63) 469 (530) P31830.1

L. ivanovii ILO 46 (43) 66 (62) 469 (528) AAR97343.1

Planococcaceae

Lysinibacillus

L. sphaericus SPH 76 (72) 90 (87) 463 (506) YP_001699692.1

Paenibacillaceae

Paenibacillus

P. alvei ALV 75 (71) 87 (84) 462 (501) P23564

Brevibacillus

B. brevis BVL 73 (69) 88 (84) 464 (511) YP_002770211.1

Lactobacillales

Streptococcaceae

Streptococcus

S. dysgalactiae* SLOe 67 (56) 83 (70) 463 (571) BAD77791

S. pyogenes SLO 67 (56) 83 (70) 463 (571) NP_268546.1

S. canis SLOc 66 (55) 82 (69) 463 (574) Q53957

S. pseudonemoniae PSY 46 (43) 67 (63) 466 (471) ACJ76900

S. pneumoniae PLY 46 (43) 67 (64) 466 (471) ABO21366.1

S. mitis MLY 46 (43) 67 (63) 466 (471) ABK58695

S. suis SLY 41 (40) 65 (63) 465 (497) ABE66337.1

S. intermedius ILY 41 (37) 65 (59) 469 (532) BAE16324.1

S. mitis (Lectinolysin) LLY 39 (29) 62 (47) 463 (665) BAE72438.1

Clostridia

Clostridiales

Clostridiaceae

Clostridium

C. perfringens PFO 463 (500) NP_561079

C. butyricum BRY 69 (65) 85 (82) 462 (513) ZP_02950902.1

C. tetani TLY 60 (55) 78 (72) 464 (527) NP_782466.1

C. botulinum B BLYb 60 (49) 78 (63) 464 (602) YP_001886995.1

C. botulinum E3 BLYe 60 (48) 77 (60) 464 (602) YP_001921918.1

C. botulinum C BLYc 60 (56) 79 (74) 463 (518) ZP_02620972.1

C. novyi NVL 58 (54) 78 (73) 463 (514) YP_878174.1

Actinobacteria

Bifidobacteriales

Bifidobacteriaceae

Gardenella

G. vaginallis VLY 40 (39) 65 (60) 466 (516) ACD3946.1

Actinomycetales

Actinomycetaceae

Arcanobacterium

A. pyogenes PLO 41 (38) 60 (56) 469 (534) AAC45754.1

FIGURE LEGENDS

Figure 1. Analysis of the primary structure for the CDC reveals a high degree of identity

and similarity among them. Only the sequence for the conserved core of the CDC was

used for the analysis (corresponding to PFO amino acids 38-500). If more than one

sequence was available for individual species, only one was used in the analysis. The

databank access numbers are provided in Table 1. Sequence relationships were calculated

using the MatGat 2.02 alignment program using the BLOSUM 62 matrix and open and

extension gap penalties of 12 and 1, respectively (Campanella et al., 2003). The identity

scores occupy the upper triangle (in bold) with scores higher than 70% shaded in dark

gray, and those at 50 – 70% in light gray. Similarity scores in the lower triangle where

shaded in dark gray if higher than 80% and in light gray if between 70 – 80%.

Figure 2. Pore formation mechanism for the CDC. Secreted as water-soluble monomeric

proteins, the toxins bind to the target membrane and oligomerize into a ring-like structure

called the pre-pore complex. A poorly understood conformational change then leads to

the insertion of the TMHs into the bilayer to form the aqueous pore. (A) Stages of PFO

pore formation. The defined PFO structural domains are numbered. The membrane

bilayer is depicted with cholesterol molecules (ovals) intercalated between the

phospholipid constituents. Membrane binding is accomplished as D4 interacts with

membrane regions having free cholesterol molecules. Subsequent allosteric

rearrangements within the monomer promote oligomerization and pore-formation. (B)

Conformational changes in domain 3 of PFO are required for monomer–monomer

association and β-barrel pore formation. Each stage corresponds to the stage shown above

in (A). The TMH1 is shown as bicolor and the TMH2 in black. The small β5 strand is

shown as a black loop. The aromatic residues involved in the alignment of the β-strands

are shown as open rectangles. Adapted from Ramachandran et al. with permission

(2004).

Figure 3. Comparison of PFO homologs reveals a conserved core backbone. Alignment

and comparison of the composite members of the CDC family reveals conserved regions

that extend from the tip of the membrane recognition domain, D4, through the regions

involved in oligomerization and membrane insertion. (A) Cartoon representation of PFO

with the conserved residues shown in black. (B) Surface representation of PFO the

conserved core highlighted in black. It is postulated that this conserved backbone is

especially adapted to allosterically communicate successful, cholesterol-dependent

membrane binding, and thus permit subsequent conformational adaptations that favor

oligomerization and pore formation. Alignment of the 28 CDC sequences was effected

using the PRALINE multiple sequence alignment tool using a BLOSUM62 matrix with

open and extension gap penalties set at 12 and 1, respectively, a PSI-BLAST pre-profile

processing with iterations set at 3, e-value cut off set at 0.01, non-redundant data bases,

and a DSSP-defined secondary structure search using PSIPRED (Simossis et al., 2005).

PFO structure representation was rendered using PyMol (DeLano Scientific LLC).

Figure 4. The three dimensional structure of D4 is highly conserved in the CDC family.

(A) Comparison of D4 from 3 CDC homologs highlights the conserved architecture of

this C-terminal domain. A cartoon, upper left, clarifies the threading of 2 β-sheets and

loops in the β-sandwich and indicates the spatial organization of the undecapeptide, L1,

and L2. The α-backbone for the D4 domains of PFO, ILY, and ALO were superimposed

using PyMol (DeLano Scientific LLC; available at www.pymol.org). (B) Alignment of

the sequence for the 28 CDC family members reveals substantial conservation in loops

L1, L2 and the undecapeptide. While integrity of the undecapeptide was long recognized

for being critical to the cholesterol-dependent activity of these toxins, other loops are also

important. Residues conserved in all sequences are shaded in black, and highly conserved

residues are shaded in gray. Protein names are as in Figure 2, residue numbers correspond

to the PFO sequence. Multiple sequence alignment was effected as indicated in Figure 3.

Figure 5. PFO only binds to membranes containing free cholesterol molecules. Examples

of mechanisms for cholesterol-dependent anchoring of PFO to the membrane surface: (A)

PFO cannot stably bind to the bilayer if there are no free cholesterol molecules available

in the membrane surface. (B) At high cholesterol concentrations free cholesterol

molecules become available (black ovals), and D4 can anchor to the bilayer. In this

example, a single cholesterol molecule binds to D4 and induces the conformational

changes required to expose the D4 loops to the bilayer core. (C) Alternatively, the

interplay between D4 and the membrane result in the redistribution of the lipids at the

surface, clustering the free cholesterol molecules underneath the tip of D4. Anchoring

may be accomplished by the interaction of multiple hydroxyl groups located in the

cholesterol-rich cluster and the conserved amino acids of the loops.

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PHYLUM

Firmicutes

CLASS

Bacilli

ORDER

Bacillales

FAMILY

Bacillaceae

GENUS

Bacillus %Identity %Similarity Length ID

SPECIES

B. anthracis ALO 72 (68) 88 (83) 462 (512) ZP_03017964.1

B. thurigiensis TLO 74 (69) 88 (83) 462 (512) YP_037419

B. cereus CLO 74 (69) 88 (84) 462 (512) YP_002369889.1

B. weihenstephanensis WLO 74 (69) 87 (83) 462 (512) ABY46062

Listeriaceae

Listeria

L. monocytogenes LLO 43 (40) 66 (62) 469 (529) DQ838568.1

L. seeligeri LSO 45 (41) 67 (63) 469 (530) P31830.1

L. ivanovii ILO 46 (43) 66 (62) 469 (528) AAR97343.1

Planococcaceae

Lysinibacillus

L. sphaericus SPH 76 (72) 90 (87) 463 (506) YP_001699692.1

Paenibacillaceae

Paenibacillus

P. alvei ALV 75 (71) 87 (84) 462 (501) P23564

Brevibacillus

B. brevis BVL 73 (69) 88 (84) 464 (511) YP_002770211.1

Lactobacillales

Streptococcaceae

Streptococcus

S. dysgalactiae* SLOe 67 (56) 83 (70) 463 (571) BAD77791

S. pyogenes SLO 67 (56) 83 (70) 463 (571) NP_268546.1

S. canis SLOc 66 (55) 82 (69) 463 (574) Q53957

S. pseudonemoniae PSY 46 (43) 67 (63) 466 (471) ACJ76900

S. pneumoniae PLY 46 (43) 67 (64) 466 (471) ABO21366.1

S. mitis MLY 46 (43) 67 (63) 466 (471) ABK58695

S. suis SLY 41 (40) 65 (63) 465

(497) ABE66337.1

S. intermedius ILY 41 (37) 65 (59) 469 (532) B212797.1

S. mitis (Lectinolysin) LLY 39 (29) 62 (47) 463 (665) BAE72438.1

Clostridia

Clostridiales

Clostridiaceae

Clostridium

C. perfringens PFO 463 (500) NP_561079

C. butyricum BRY 69 (65) 85 (82) 462 (513) ZP_02950902.1

C. tetani TLY 60 (55) 78 (72) 464 (527) NP_782466.1

C. botulinum B BLYb 60 (49) 78 (63) 464 (602) YP_001886995.1

C. botulinum E3 BLYe 60 (48) 77 (60) 464 (602) YP_001921918.1

C. botulinum C BLYc 60 (56) 79 (74) 463 (518) ZP_02620972.1

C. novyi NVL 58 (54) 78 (73) 463 (514) YP_878174.1

Actinobacteria

Bifidobacteriales

Bifidobacteriaceae

Gardenella

G. vaginallis VLY 40 (39) 65 (60) 466 (516) EU522488.1

Actinomycetales

Actinomycetaceae

Arcanobacterium

A. pyogenes PLO 41 (38) 60 (56) 469 (534) U84782.2

Perfringolysin O

76 72 74 74 74 75 73 69 60 60 60 60 58 67 66 67 39 46 46 46 41 41 46 45 43 40 41

Sphaericolysin

SPH 90 80 82 82 82 77 80 69 58 57 57 56 54 66 65 65 37 42 42 42 41 37 45 45 43 37 38

Anthrolysin O

ALO 88 93 97 97 97 73 76 65 57 56 56 58 55 62 61 62 37 41 42 42 40 39 44 43 41 41 39

Cereolysin O

CLO 88 93 99 98 98 75 76 67 57 57 57 58 54 63 62 63 37 41 41 41 40 39 44 43 42 40 39

Thuringiensilysin O

TLO 88 93 99 99 98 75 77 66 57 57 57 57 54 63 62 63 37 41 41 42 40 39 44 43 42 40 39

Weihenstephanensilysin

WLO 87 93 99 99 99 75 77 66 56 57 57 57 54 63 62 63 37 41 41 42 40 39 43 43 42 40 39

Alveolysin

ALV 87 89 88 88 88 88 72 65 60 57 58 57 53 64 63 64 39 41 41 41 40 40 44 43 42 40 39

Brevilysin

BVL 88 93 91 91 92 92 87 68 58 57 57 58 57 64 63 64 39 42 42 42 40 39 46 44 42 39 40

Butyriculysin

BRY 85 86 84 84 84 84 81 84 58 55 55 57 56 64 63 63 37 43 43 43 40 39 45 44 42 40 39

Tetanolysin O

TLY 78 79 77 78 77 77 77 76 75 82 81 81 77 55 55 55 42 45 45 45 45 42 50 50 48 43 44

Botulinolysin B

BLYb 77 77 75 76 76 75 75 75 74 93 95 77 73 55 55 55 42 46 46 46 45 43 51 49 48 43 44

Botulinolysin E3

BLYe 77 77 75 75 75 75 75 74 75 93 98 74 72 54 54 54 42 44 45 44 45 42 51 49 49 43 43

Botulinolysin C

BLYc 79 79 78 79 78 78 77 77 77 93 90 90 83 54 54 54 42 46 46 47 46 46 49 49 48 45 43

Novyilysin

NVL 77 78 79 79 79 79 76 77 78 90 88 87 93 53 52 52 42 44 44 45 45 44 50 50 47 44 44

Streptolysin O

SLO 83 81 79 80 80 79 80 81 80 74 73 73 74 75 99 100 39 42 42 42 41 38 43 43 41 39 39

Streptolysin O c

SLOc 82 80 79 79 79 79 79 81 79 74 73 72 74 74 99 99 39 41 41 41 40 37 44 43 42 39 39

Streptolysin O e

SLOe 83 81 79 80 80 79 80 81 80 74 73 73 74 75 100 99 39 41 41 42 41 37 44 43 42 39 39

Lectinolysin

LLY 62 61 61 61 61 61 62 62 63 65 64 64 66 64 64 63 64 51 51 51 46 54 40 41 40 59 41

Pneumolysin

PLY 67 66 65 66 66 65 66 66 66 68 67 67 69 67 65 64 65 73 99 99 51 53 43 43 43 53 41

Mitilysin

MLY 67 66 65 66 66 65 66 66 66 67 67 67 69 67 65 64 65 73 100 100 50 53 44 43 43 53 41

Pseudopneumolysin

PSY 67 66 65 66 66 65 66 66 66 67 67 67 69 67 65 64 65 73 100 100 50 53 44 43 43 53 40

Suilysin

SLY 65 65 66 66 66 65 65 66 66 69 68 68 69 69 65 64 65 68 73 74 74 47 48 45 46 52 45

Intermedilysin

ILY 64 62 64 64 64 64 64 63 64 65 66 66 68 67 62 61 62 77 74 74 74 68 42 42 42 60 42

Ivanolysin

ILO 66 64 65 64 64 64 64 66 66 70 70 70 71 71 65 65 65 64 67 67 67 71 66 79 81 42 43

Seeligeriolysin O

LSO 67 66 65 65 66 65 67 67 65 71 70 71 71 70 66 66 66 64 66 66 66 69 64 91 85 41 43

Listeriolysin O

LLO 66 65 63 64 64 63 65 66 64 72 71 71 71 70 66 66 66 65 67 67 67 69 66 94 95 40 43

Vaginolysin

VLY 63 62 64 64 64 64 63 62 62 64 65 64 66 67 62 61 62 77 73 73 73 69 79 63 62 63 43

Pyolysin

PLO 60 58 58 59 59 59 60 61 61 61 60 60 63 63 60 60 60 62 61 61 61 64 63 63 62 62 60

PFO

CLO

ALO

SPH

PLO

VLY

LLO

LSO

ILO

ILY

SLY

PSY

MLY

PLY

LLY

SLOe

SLOc

SLO

NVL

BLYc

BLYe

BLYb

TLY

BRY

BVL

ALV

WLO

TLO

PFO

II III

β1

β2β3

β4

β5

conserved loops

domain 4

ILY

PFO

ACOOH

undecapeptide

PFO H S G A Y V A

FDKTAHYECTGLAWEWWRGTTLYP

ALO H Y G A Y V A

FDKTAHYECTGLAWEWWRGTTLYP

CLO H Y G A Y V A

FDKTAHYECTGLAWEWWRGTTLYP

WLO H Y G A Y V A

FDKTAHYECTGLAWEWWRGTTLYP

TLO H Y G A Y V A

FDKTAHYECTGLAWEWWRGTTLYP

SPH H Y G A Y V A

FDKTAHFECTGLAWEWWRGTTLYP

BLYe H S G A Y V A

FDKTAHFECTGLAWEWWRGTTLYP

BLYb H S G A Y V A

FDKTAHFECTGLAWEWWRGTTLYP

ALV H S G A Y V A

FDRSAHFECTGLAWEWWRGTTLYP

TLY H S G A Y V A

FDRTAHFECTGLAWEWWRGTTLYP

NVL H R G A Y V A K F G R T A H F E C T G L A W E W W R G T T L Y P

BLYc H S G A Y V A

FDRTAHFECTGLAWEWWRGTTLYP

ILO HSGAYVARF DKLAHF ECTGLAWEWWR GTTLYP

LLO H S G G Y V A

FSKLAHFECTGLAWEWWRGTTLYP

PLY H S G A Y V A

YDLTAHFECTGLAWEWWRGTTLYP

MLY H S G A Y V A

YDLTAHFECTGLAWEWWRGTTLYP

PSY H S G A Y V A

YDLTAHFECTGLAWEWWRGTTLYP

SLY H S G A Y V A K Y N L T S H W E C T G L A W E W W R G T T L Y P

SLO H

GAYVA

Y SKTSPF ECTGLAWEWWR GSTLSP

SLOc H

GAYVA

Y SKTSPF ECTGLAWEWWR GSTLSP

SLOe H

GAYVA

Y SKTSPF ECTGLAWEWWR GSTLSP

BVL H Y G W Y V A

FDRTAPFECTGLAWEWWRGTTLNP

LSO H S G G Y V A

FSKLAHFECTGLFWEWWRGTTLYP

BRY H Y G A Y V A

FDKTAHFECTGLSWEWWRGTTLYP

VLY H R G A Y V A R Y Y R T A H F E K T G L V W E P W R G T T L W P

LLY H K G A Y V A R Y N R T S G F E K T G L V W E P W R G T T L N P

ILY H D G A F V A R F N R G A H Y G A T G L A W E P W R G T T L H P

PLO HGGGYVAKF ARTLGF EATGLAWDPWW GTT LNP

398-406

434-439

undecapeptide

458-468

488-493

ALO

undecapeptide

COOH

Revealing the Mechanism of NLRP3 Inflammatory Pathway Activation through K+ Efflux Induced by PLO via Signal Point Mutations

Article

Full-text available

Jun 2024
INT J MOL SCI

Trueperella pyogenes is an important opportunistic pathogenic bacterium widely distributed in the environment. Pyolysin (PLO) is a primary virulence factor of T. pyogenes and capable of lysing many different cells. PLO is a member of the cholesterol-dependent cytolysin (CDC) family of which the primary structure only presents a low level of homology with other members from 31% to 45%. By deeply studying PLO, we can understand the overall pathogenic mechanism of CDC family proteins. This study established a mouse muscle tissue model infected with recombinant PLO (rPLO) and its single-point mutations, rPLO N139K and rPLO F240A, and explored its mechanism of causing inflammatory damage. The inflammatory injury abilities of rPLO N139K and rPLO F240A are significantly reduced compared to rPLO. This study elaborated on the inflammatory mechanism of PLO by examining its unit point mutations in detail. Our data also provide a theoretical basis and practical significance for future research on toxins and bacteria.

Unique features of β-cell metabolism are lost in type 2 diabetes

Article

Full-text available

Apr 2024
ACTA PHYSIOL

Pancreatic β cells play an essential role in the control of systemic glucose homeostasis as they sense blood glucose levels and respond by secreting insulin. Upon stimulating glucose uptake in insulin‐sensitive tissues post‐prandially, this anabolic hormone restores blood glucose levels to pre‐prandial levels. Maintaining physiological glucose levels thus relies on proper β‐cell function. To fulfill this highly specialized nutrient sensor role, β cells have evolved a unique genetic program that shapes its distinct cellular metabolism. In this review, the unique genetic and metabolic features of β cells will be outlined, including their alterations in type 2 diabetes (T2D). β cells selectively express a set of genes in a cell type‐specific manner; for instance, the glucose activating hexokinase IV enzyme or Glucokinase ( GCK ), whereas other genes are selectively “disallowed”, including lactate dehydrogenase A ( LDHA ) and monocarboxylate transporter 1 ( MCT1 ). This selective gene program equips β cells with a unique metabolic apparatus to ensure that nutrient metabolism is coupled to appropriate insulin secretion, thereby avoiding hyperglycemia, as well as life‐threatening hypoglycemia. Unlike most cell types, β cells exhibit specialized bioenergetic features, including supply‐driven rather than demand‐driven metabolism and a high basal mitochondrial proton leak respiration. The understanding of these unique genetically programmed metabolic features and their alterations that lead to β‐cell dysfunction is crucial for a comprehensive understanding of T2D pathophysiology and the development of innovative therapeutic approaches for T2D patients.

Inhibition of Carbohydrate Metabolism Potentiated by the Therapeutic Effects of Oxidative Phosphorylation Inhibitors in Colon Cancer Cells

Article

Full-text available

Apr 2024

Simple Summary Glycolysis and oxidative phosphorylation play important roles in the progression and growth of cancers. The development of natural products and their semisynthetic derivatives for cancer treatment is a longstanding focus of our research interests. We developed compounds known as diaminobutoxy-substituted isoflavonoids (DBIs) that effectively stimulated Adenosine 5′ Monophosphate-activated Protein Kinase (AMPK) and suppressed the growth of colorectal cancer cells by specifically targeting mitochondrial complex I. We now report a new DBI analog, namely, DBI-2, with promising properties for cancer treatment. The combination of DBI-2 and BAY-876, a glucose transporter 1 inhibitor, exhibited synergistic effects on colorectal cancer cells. Furthermore, the therapeutic effectiveness of DBI-2 in colorectal cancer cell xenograft mouse models was enhanced by implementing a ketogenic diet, an outcome that indicated this drug/diet combination is a potentially promising combination strategy for cancer therapy. Abstract Cancer cells undergo a significant level of “metabolic reprogramming” or “remodeling” to ensure an adequate supply of ATP and “building blocks” for cell survival and to facilitate accelerated proliferation. Cancer cells preferentially use glycolysis for ATP production (the Warburg effect); however, cancer cells, including colorectal cancer (CRC) cells, also depend on oxidative phosphorylation (OXPHOS) for ATP production, a finding that suggests that both glycolysis and OXPHOS play significant roles in facilitating cancer progression and proliferation. Our prior studies identified a semisynthetic isoflavonoid, DBI-1, that served as an AMPK activator targeting mitochondrial complex I. Furthermore, DBI-1 and a glucose transporter 1 (GLUT1) inhibitor, BAY-876, synergistically inhibited CRC cell growth in vitro and in vivo. We now report a study of the structure–activity relationships (SARs) in the isoflavonoid family in which we identified a new DBI-1 analog, namely, DBI-2, with promising properties. Here, we aimed to explore the antitumor mechanisms of DBIs and to develop new combination strategies by targeting both glycolysis and OXPHOS. We identified DBI-2 as a novel AMPK activator using an AMPK phosphorylation assay as a readout. DBI-2 inhibited mitochondrial complex I in the Seahorse assays. We performed proliferation and Western blotting assays and conducted studies of apoptosis, necrosis, and autophagy to corroborate the synergistic effects of DBI-2 and BAY-876 on CRC cells in vitro. We hypothesized that restricting the carbohydrate uptake with a KD would mimic the effects of GLUT1 inhibitors, and we found that a ketogenic diet significantly enhanced the therapeutic efficacy of DBI-2 in CRC xenograft mouse models, an outcome that suggested a potentially new approach for combination cancer therapy.

Flanking N- and C-terminal domains of PrsA in Streptococcus suis type 2 are crucial for inducing cell death independent of TLR2 recognition

Article

Full-text available

Aug 2023

Streptococcus suis type 2 (SS2), a major emerging/re-emerging zoonotic pathogen found in humans and pigs, can cause severe clinical infections, and pose public health issues. Our previous studies recognized peptidyl-prolyl isomerase (PrsA) as a critical virulence factor promoting SS2 pathogenicity. PrsA contributed to cell death and operated as a pro-inflammatory effector. However, the molecular pathways through which PrsA contributes to cell death are poorly understood. Here in this study, we prepared the recombinant PrsA protein and found that pyroptosis and necroptosis were involved in cell death stimulated by PrsA. Specific pyroptosis and necroptosis signalling inhibitors could significantly alleviate the fatal effect. Cleaved caspase-1 and IL-1β in pyroptosis with phosphorylated MLKL proteins in necroptosis pathways, respectively, were activated after PrsA stimulation. Truncated protein fragments of enzymatic PPIase domain (PPI), N-terminal (NP), and C-terminal (PC) domains fused with PPIase, were expressed and purified. PrsA flanking N- or C-terminal but not enzymatic PPIase domain was found to be critical for PrsA function in inducing cell death and inflammation. Additionally, PrsA protein could be anchored on the cell surface to interact with host cells. However, Toll-like receptor 2 (TLR2) was not implicated in cell death and recognition of PrsA. PAMPs of PrsA could not promote TLR2 activation, and no rescued phenotypes of death were shown in cells blocking of TLR2 receptor or signal-transducing adaptor of MyD88. Overall, these data, for the first time, advanced our perspective on PrsA function and elucidated that PrsA-induced cell death requires its flanking N- or C-terminal domain but is dispensable for recognizing TLR2. Further efforts are still needed to explore the precise molecular mechanisms of PrsA-inducing cell death and, therefore, contribution to SS2 pathogenicity.

Genomic and pathogenicity islands of Listeria monocytogenes—overview of selected aspects

Article

Full-text available

Jun 2023

Listeria monocytogenes causes listeriosis, a disease characterized by a high mortality rate (up to 30%). Since the pathogen is highly tolerant to changing conditions (high and low temperature, wide pH range, low availability of nutrients), it is widespread in the environment, e.g., water, soil, or food. L. monocytogenes possess a number of genes that determine its high virulence potential, i.e., genes involved in the intracellular cycle (e.g., prfA , hly , plcA , plcB , inlA , inlB ), response to stress conditions (e.g., sigB , gadA , caspD , clpB , lmo1138 ), biofilm formation (e.g., agr, luxS ), or resistance to disinfectants (e.g., emrELm , bcrABC , mdrL ). Some genes are organized into genomic and pathogenicity islands. The islands LIPI-1 and LIPI-3 contain genes related to the infectious life cycle and survival in the food processing environment, while LGI-1 and LGI-2 potentially ensure survival and durability in the production environment. Researchers constantly have been searching for new genes determining the virulence of L. monocytogenes . Understanding the virulence potential of L. monocytogenes is an important element of public health protection, as highly pathogenic strains may be associated with outbreaks and the severity of listeriosis. This review summarizes the selected aspects of L. monocytogenes genomic and pathogenicity islands, and the importance of whole genome sequencing for epidemiological purposes.

A basic model for the association of ligands with membrane cholesterol: application to cytolysin binding

Article

Full-text available

Feb 2023
J LIPID RES

Almost all the cholesterol in cellular membranes is associated with phospholipids in simple stoichiometric complexes. This limits the binding of sterol ligands such as filipin and Perfringolysin O (PFO) to a small fraction of the total. We offer a simple mathematical model that characterizes this complexity. It posits that the cholesterol accessible to ligands has two forms: active cholesterol, which is that not complexed with phospholipids; and extractable cholesterol, that which ligands can capture competitively from the phospholipid complexes. Simulations based on the model match published data for the association of PFO oligomers with liposomes, plasma membranes and the isolated endoplasmic reticulum. The model shows how the binding of a probe greatly underestimates cholesterol abundance when its affinity for the sterol is so weak that it competes poorly with the membrane phospholipids. Two examples are the under-staining of plasma membranes by filipin and the failure of domain D4 of PFO to label their cytoplasmic leaflets. Conversely, the exaggerated staining of endolysosomes suggests that their cholesterol, being uncomplexed, is readily available. The model is also applicable to the association of cholesterol with intrinsic membrane proteins. For example, it supports the hypothesis that the sharp threshold in the regulation of homeostatic ER proteins by cholesterol derives from the cooperativity of their binding to the sterol weakly held by the phospholipid. § Thus, the model explicates the complexity inherent in the binding of ligands like PFO and filipin to the small accessible fraction of membrane cholesterol.

Overview of Bacterial Protein Toxins from Pathogenic Bacteria: Mode of Action and Insights into Evolution

Article

Full-text available

Apr 2024

Michel Popoff

Bacterial protein toxins are secreted by certain bacteria and are responsible for mild to severe diseases in humans and animals. They are among the most potent molecules known, which are active at very low concentrations. Bacterial protein toxins exhibit a wide diversity based on size, structure, and mode of action. Upon recognition of a cell surface receptor (protein, glycoprotein, and glycolipid), they are active either at the cell surface (signal transduction, membrane damage by pore formation, or hydrolysis of membrane compound(s)) or intracellularly. Various bacterial protein toxins have the ability to enter cells, most often using an endocytosis mechanism, and to deliver the effector domain into the cytosol, where it interacts with an intracellular target(s). According to the nature of the intracellular target(s) and type of modification, various cellular effects are induced (cell death, homeostasis modification, cytoskeleton alteration, blockade of exocytosis, etc.). The various modes of action of bacterial protein toxins are illustrated with representative examples. Insights in toxin evolution are discussed.

Dual-monomer solvatochromic probe system (DSPS) for effectively differentiating lipid raft cholesterol and active membrane cholesterol in the inner-leaflet plasma membrane

Article

Feb 2024

A novel, selective, practical cholesterol sensing system based on a fusion protein and a solvatochromic molecule was developed to distinguish cholesterol exposed in different phases of the plasma membrane.

CD4+ T cell-mediated recognition of a conserved cholesterol-dependent cytolysin epitope generates broad antibacterial immunity

Article

Apr 2023
IMMUNITY

CD4+ T cell-mediated immunity against Streptococcus pneumoniae (pneumococcus) can protect against recurrent bacterial colonization and invasive pneumococcal diseases (IPDs). Although such immune responses are common, the pertinent antigens have remained elusive. We identified an immunodominant CD4+ T cell epitope derived from pneumolysin (Ply), a member of the bacterial cholesterol-dependent cytolysins (CDCs). This epitope was broadly immunogenic as a consequence of presentation by the pervasive human leukocyte antigen (HLA) allotypes DPB1∗02 and DPB1∗04 and recognition via architecturally diverse T cell receptors (TCRs). Moreover, the immunogenicity of Ply427-444 was underpinned by core residues in the conserved undecapeptide region (ECTGLAWEWWR), enabling cross-recognition of heterologous bacterial pathogens expressing CDCs. Molecular studies further showed that HLA-DP4-Ply427-441 was engaged similarly by private and public TCRs. Collectively, these findings reveal the mechanistic determinants of near-global immune focusing on a trans-phyla bacterial epitope, which could inform ancillary strategies to combat various life-threatening infectious diseases, including IPDs.

Molecular mechanism of Streptococcus pneumoniae –targeting xenophagy recognition and evasion: Reinterpretation of pneumococci as intracellular bacteria

Article

Mar 2023

Streptococcus pneumoniae is a major, encapsulated gram‐positive pathogen that causes diseases including community‐acquired pneumonia, meningitis, and sepsis. This pathogen colonizes the nasopharyngeal epithelia asymptomatically but can often migrate to sterile tissues and cause life‐threatening invasive infections (invasive pneumococcal disease: IPD). Although multivalent pneumococcal polysaccharides and conjugate vaccines are available and effective, they also have major shortcomings with respect to the emergence of vaccine‐resistant serotypes. Therefore, alternative therapeutic approaches are needed, and the molecular analysis of host‐pathogen interactions and their applications to pharmaceutical development and clinical practice has recently received increased attention. In this review, we introduce pneumococcal surface virulence factors involved in pathogenicity and highlight recent advances in understanding regarding host autophagy recognition mechanisms against intracellular S. pneumoniae and pneumococcal evasion from autophagy. This article is protected by copyright. All rights reserved.

Membrane Recognition and Pore Formation by Bacterial Pore-forming Toxins

Article

Mar 2006

IntroductionClassification of Bacterial PFTs α-PFTsβ-PFTsA General Mechanism of Pore Formation?Membrane Recognition Recognition of Specific Membrane LipidsRecognition of Membrane-anchored Proteins or CarbohydratesThe Role of Membrane Lipid DomainsOligomerization on the Membrane Surface Oligomerization Triggered by Lipid-induced Conformational ChangesOligomerization Following Proteolytic Activation of ToxinsMembrane Penetration and Pore FormationUnresolved IssuesReferences α-PFTsβ-PFTs Recognition of Specific Membrane LipidsRecognition of Membrane-anchored Proteins or CarbohydratesThe Role of Membrane Lipid Domains Oligomerization Triggered by Lipid-induced Conformational ChangesOligomerization Following Proteolytic Activation of Toxins

Repertoire and general features of the family of cholesterol-dependent cytolysins

Chapter

Dec 2006

The cholesterol-dependent cytolysins (CDCs), also called cholesterol-binding cytolysins, constitute a group of 50- to 60-kDa single-chain, pore-forming bacterial protein toxins previously designated "sulfhydryl" (or "thiol-activated)" cytolysins. This group of structurally, antigenically, and functionally related cytolysins constitutes the largest family of bacterial protein toxins produced by Gram-positive bacteria. CDCs are lethal to animals and highly lytic towards eucaryotic cells, including erythrocytes. Their lytic and lethal properties are suppressed by sulfhydryl-group blocking agents and reversibly restored by thiols or other reducing agents. This chapter classifies and lists the general features of this family of cholesterol-dependant cytolysins. Important progress has been made in the past five years with regard to the mechanisms of toxin insertion and oligomerization in target cells in relation to membrane cholesterol and the genetics and structural characteristics of these toxins. However, the role of certain CDCs in disease, particularly in inflammatory processes and probably in sepsis, remains poorly understood. Moreover, the involvement of CDCs in host-signaling pathways still requires further investigation. Finally, the potential use of certain CDCs as potent permeabilization probes in cell biology or as vaccines or novel therapeutical agents remains an important and promising issue.

Condensed complexes of cholesterol and phospholipids

Article

Mar 2003
BBA-BIOMEMBRANES

HM McConnell

There is overwhelming evidence that lipid bilayer regions of animal cell membranes are in a liquid state. Quantitative models of these bilayer regions must then be models of liquids. These liquids are highly non-ideal. For example, it has been known for more than 75 years that mixtures of cholesterol and certain phospholipids undergo an area contraction or condensation in lipid monolayers at the air–water interface. In the past 3 years, a thermodynamic model of “condensed complexes” has been proposed to account for this non-ideal behavior. Here we give an overview of the model, its relation to other models, and to modern views of the properties of animal cell membranes.

Electron microscopy of cholesterol

Article

Dec 1988

James Robin Harris

Negative staining and platinum-carbon shadowing have been used to prepare electron microscope specimens from aqueous colloidal suspensions of cholesterol microscrystals and from crystalline suspensions in methanol and ethanol. Microcrystals prepared by injection of alcoholic solutions of cholesterol into water exhibit angular conformations of varying regularity which contain a number of parallel cholesterol bilayers. The electron optical images of the cholesterol microcrystals, oriented horizontally and ‘on-edge’, obtained by both negative staining and metal shadowing, are in good agreement. Metal shadowing does, however, reveal greater detail within microcrystal clusters than does negative staining, as well as of the bilayer steps at microcrystal edges. The needle-like crystals (from methanol) and plate-like crystals (from ethanol) present considerable difficulties for the negative staining technique, because of their thickness and the consequent depth of the surrounding negative stain. Small crystals are, nevertheless, shown to possess multiple cholesterol bilayers. Platinum-carbon shadowing of cholesterol crystals taken directly from methanol and ethanol provides more satisfactory images than negative staining. The large depth of focus of the transmission electron microscope enables the stacked cholesterol bilayers to be clearly defined at the edges of crystals. The results obtained are discussed in relation to the physicochemical and biological properties of cholesterol, which underlie the fundamental difficulty encountered when fixing and staining cholesterol for thin sectioning, and also the role of cholesterol insolubility in the formation of gallstones.

Scientific Books: Immunochemistry. The Application of the Principles of Physical Chemistry to the Study of the Biological Antibodies

Article

Jan 1908

Svante Arrhenius

β-Barrel PoreForming Toxins: Intriguing Dimorphic Proteins †

Article

Aug 2001

The Mechanism of Pore Assembly for a Cholesterol-Dependent Cytolysin: Formation of a Large Prepore Complex Precedes the Insertion of the Transmembrane β-Hairpins †

Article

Aug 2000

Perfringolysin O (PFO) is a member of the cholesterol-dependent cytolysin (CDC) family of membrane-penetrating toxins. The CDCs form large homooligomers (estimated to be comprised of up to 50 CDC monomers) that are responsible for generating a large pore in cholesterol-containing membranes of eukaryotic cells. The assembly of the PFO cytolytic complex was examined to determine whether it forms an oligomeric prepore complex on the membrane prior to the insertion of its membrane-spanning beta-sheet. A PFO oligomeric complex was formed on liposomes at both 4 degrees C and 37 degrees C and shown by SDS-agarose gel electrophoresis to be comprised of a large, comparatively homogeneous complex instead of a distribution of oligomer sizes. At low temperature, the processes of oligomerization and membrane insertion could be resolved, and PFO was found to form an oligomer without significant membrane insertion of its beta-hairpins. Furthermore, PFO was found to increase the ion conductivity through a planar bilayer by large and discrete stepwise changes in conductance that are consistent with the insertion of a preassembled pore complex into the bilayer. The combined results of these analyses strongly support the hypothesis that PFO forms a large oligomeric prepore complex on the membrane surface prior to the insertion of its transmembrane beta-sheet.

Interaction of .theta.Toxin (Perfringolysin O), a Cholesterol-Binding Cytolysin, with Liposomal Membranes: Change in the Aromatic Side Chains upon Binding and Insertion

Article

May 1995

TO understand the mechanism of membrane lysis by theta-toxin (perfringolysin O) from Clostridium perfringens, a cholesterol-binding, pore-forming cytolysin, we undertook a spectroscopic analysis of the structural changes that occur during the lytic process using lipid vesicles. In particular, the spectra were compared with those obtained using a modified theta-toxin, MC theta, that binds membrane cholesterol without forming oligomeric pores, thus bypassing the oligomerization step. The interaction of theta-toxin liposomes composed of cholesterol and phosphatidylcholine but not with cholesterol-free liposomes caused a remarkable increase in the intensity of the tryptophan fluorescence emission spectra and ellipticity changes in the near- and far-UV CD peaks. A CD peak shift from 292 to 300 nm was specific for theta-toxin, suggesting oligomerization-specific changes occurring around tryptophan residues. Structural changes in the aromatic side chains were detected in the near-UV CD and fluorescence spectra upon MC theta-liposome interaction, although the far-UV CD spectra indicate that the beta-rich secondary structure of MC theta is well-conserved after membrane binding. Quenching of the intrinsic tryptophan fluorescence of MC theta by brominated lecithin/cholesterol liposomes suggests that theta-toxin inserts at least partly into membranes in the absence of oligomerization. These results indicate that regardless of oligomerization, the binding of theta-toxin to cholesterol induces partial membrane insertion and triggers conformational changes accompanied by aromatic side chain rearrangement with retention of secondary structure. The spectral changes depend on the cholesterol/toxin molar ratio and pH, with maxima at pH 5-7, correlating with the optima for binding, suggesting that the cholesterol-induced insertion mechanism is distinct from the acid-induced one.

Mechanism of Membrane Insertion of a Multimeric β-Barrel Protein

Article

Nov 2000
MOL CELL

Perfringolysin O, a bacterial cytolytic toxin, forms unusually large pores in cholesterol-containing membranes by the spontaneous insertion of two of its four domains into the bilayer. By monitoring the kinetics of domain-specific conformational changes and pore formation using fluorescence spectroscopy, the temporal sequence of domain-membrane interactions has been established. One membrane-exposed domain does not penetrate deeply into the bilayer and is not part of the actual pore, but is responsible for membrane recognition. This domain must bind to the membrane before insertion of the other domain into the bilayer is initiated. The two domains are conformationally coupled, even though they are spatially separated. Thus, cytolytic pore formation is accomplished by a novel mechanism of ordered conformational changes and interdomain communication.

The projection structure of Perfringolysin O (Clostridium perfringens θ-toxin)

Article

Mar 1993

The cytolysin Perfringolysin O was applied to lipid layers and the obtained ring-shaped oligomers analyzed by electron microscopy and image processing. The final result shows the periodic repeat of 2.4 nm along the outer rim of the ring. The asymmetric protein unit, corresponding to one monomer, spans the ring from the convex to the concave surface. It shows a clear protein peak close to the outer radius and less density in the middle of the oligomer. The number of monomers in the average ring is 50, and the inner radius of the aggregate is approximately 15 nm.

The Cholesterol-Dependent Cytolysin Family of Gram-Positive Bacterial Toxins

Abstract

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