**3.1 Exposure of intrinsically disordered regions**

After the initial activation of HlyA by acylation, the toxin is exported into the extracellular medium by the type-I secretion system and by OMVs. None of the secretion routes are acylation-dependent, although the extracellular transport yield was found to be lower for proHlyA compared to that for HlyA. In addition, a high concentration of ProHlyA was found in inclusion bodies (Sanchez-Magraner *et al.*, 2006). For comparative studies where acylated and nonacylated proteins were used, proHlyA was obtained from *E. coli* DH1—it having been transformed by a recombinant plasmid, pSF4000∆*BamHI*, in whose DNA a portion of the *hlyC* gene had been deleted. This strain secreted a full-length, but inactive hemolysin. Fatty acids were not necessary for the secretion of the toxin by OMVs, or by the bacteria's own export machinery; but they were essential for the toxin's hemolytic activity (Boehm *et al.*, 1990).

Several steps are involved in the lytic mechanism of the toxin: a binding of calcium previous to the toxin's interaction with membranes, the binding to and insertion into membranes, and the oligomerization of the toxin to form the final lytic pore. We will discuss below to what extent covalently bound fatty acids influence the different steps.

In the extracellular medium, HlyA must associate with calcium in order to bind to membranes in the lytically active form (Ostolaza & Goñi 1995), (Bakás *et al.*, 1998). This second activation step is acylation-dependent because the calcium-binding capacity is lower in the unacylated protein (Soloaga *et al.,* 1996). Once HlyA is calcium-activated, the toxin appears to have a two-stage interaction with membranes: first, a reversible adsorption that is sensitive to electrostatic forces; and second, an irreversible insertion (Bakás *et al.*, 1996). The inserted HlyA behaves as an integral protein because this form of the toxin cannot be extracted without the use of detergents (Soloaga *et al.*, 1999).

acetylation patterns (Hamon *et al.,* 2007), (Ratner *et al.*, 2006). In addition, sublytic concentrations of HlyA have been recently found to potently stimulate the inactivation of the serine/threonine protein kinase B (PKB), which enzyme plays a central role in host cellcycle progression, metabolism, vesicular trafficking, survival, and inflammatory-signaling pathways (Wiles *et al.*, 2008). These findings may help to explain previously published results implicating sublytic concentrations of HlyA in the inhibition of chemotaxis and in bacterial killing by phagocytes in addition to the HlyA-mediated stimulation of host apoptotic and inflammatory pathways (Cavalieri & Snyder, 1982), (Koschinski *et al.*, 2006),

In general, lipid moieties play central roles in protein function—*e. g.*, the targeting into membranes, an increase in the affinity for biological membranes, and an enhancement of

After a brief introduction to the general aspects of HlyA in the following section, we will describe the role that covalently bound fatty acids play in the mechanism of action of the toxin, from its initial activation to its final functioning in the target cell. This posttranslational modification must be critical since the presence of fatty acids transforms

After the initial activation of HlyA by acylation, the toxin is exported into the extracellular medium by the type-I secretion system and by OMVs. None of the secretion routes are acylation-dependent, although the extracellular transport yield was found to be lower for proHlyA compared to that for HlyA. In addition, a high concentration of ProHlyA was found in inclusion bodies (Sanchez-Magraner *et al.*, 2006). For comparative studies where acylated and nonacylated proteins were used, proHlyA was obtained from *E. coli* DH1—it having been transformed by a recombinant plasmid, pSF4000∆*BamHI*, in whose DNA a portion of the *hlyC* gene had been deleted. This strain secreted a full-length, but inactive hemolysin. Fatty acids were not necessary for the secretion of the toxin by OMVs, or by the bacteria's own export machinery; but they were essential for the toxin's hemolytic

Several steps are involved in the lytic mechanism of the toxin: a binding of calcium previous to the toxin's interaction with membranes, the binding to and insertion into membranes, and the oligomerization of the toxin to form the final lytic pore. We will discuss below to what

In the extracellular medium, HlyA must associate with calcium in order to bind to membranes in the lytically active form (Ostolaza & Goñi 1995), (Bakás *et al.*, 1998). This second activation step is acylation-dependent because the calcium-binding capacity is lower in the unacylated protein (Soloaga *et al.,* 1996). Once HlyA is calcium-activated, the toxin appears to have a two-stage interaction with membranes: first, a reversible adsorption that is sensitive to electrostatic forces; and second, an irreversible insertion (Bakás *et al.*, 1996). The inserted HlyA behaves as an integral protein because this form of the toxin cannot be extracted without the use of detergents (Soloaga *et al.*, 1999).

(Mansson *et al.,* 2007), (Tran Van Nhieu *et al.*,2004), (Uhlen *et al.*, 2000).

protein-protein interactions (Stanley *et al.*, 1998), (Chow *et al*., 1992).

extent covalently bound fatty acids influence the different steps.

**3. Role of the fatty acids covalently bound to HlyA** 

the innocuous proHlyA into the virulent toxin HlyA.

**3.1 Exposure of intrinsically disordered regions** 

activity (Boehm *et al.*, 1990).

Nevertheless, proHlyA, though nonacylated, also interacts with membranes. This observation is not surprising because the amino-acid sequence of the polypeptide shows amphipathic helices in the 250–400 amino-acid region. Despite the amphipathic stretches known to be essential for lytic activity, however, proHlyA is unable to alter the bilayer permeability (Soloaga *et al.*, 1999). Experiments on protein adsorption at an air-water interface suggested that the fatty acids present in HlyA, unlike those in proHlyA, did not modify the surface-active properties of the protein and that the main difference between the precursor and the mature protein was that the proHlyA was virtually unable to insert itself into lipid monolayers (Sanchez-Magraner *et al.*, 2006). Furthermore, we found that the presence of two acyl chains in HlyA confers on this protein the property of irreversible binding to membranes, which feature is essential for the lytic process to take place (Herlax & Bakas, 2003). In summary, although fatty acids covalently bound to HlyA help the toxin to bind calcium in order to adopt a competitive conformation for interaction with membranes, the absence of these fatty acids does not modify that interaction of the toxin, so that these fatty acids must play some other relevant role. The answer is that the fatty acids expose intrinsically disordered regions of the toxin that are involved in a different step within the mechanism of action.

HlyA has a molten-globule conformation promoted by the presence of acyl chains, as demonstrated by a lower denaturing concentration of guanidinium-chloride. Other characteristics demonstrating this conformation were the binding of a higher number of 8 anilinonaphtalene-1-sulfonate (ANS) molecules to HlyA with a weaker affinity, a higher efficiency of energy transfer from tryptophan to the bound ANS, and a faster digestion of HlyA with trypsin compared to the same reactions with proHlyA (Herlax & Bakas, 2007).

The acylated protein was more stable in the absence of denaturant than the unacylated form, as demonstrated by the higher ∆*G*°H2O value for HlyA compared to proHlyA. Acyl chains covalently bound to the protein, however, promote a steric hindrance that contributes to a more relaxed structure, which acylated form can thus be denatured at a lower guanidiniumchloride concentration.

ANS binding to ordered regions can be distinguished from the binding to molten-globulelike regions by differences in the apparent binding constant. The exceptionally high value of ANS bound to HlyA and proHlyA might result from amphipathic regions in both forms, but the presence of fatty acids has been observed to double this value because of the molten structure those lipids impart. The binding of a large number of ANS molecules in a weak manner is characteristic of the loose structure of the molten conformation. ANS binding to pockets in ordered or molten-globule proteins operationally gives apparent *K*d values that differ by more than a factor of 5; thus, despite the uncertainties involved, these apparent *K*d values serve as a diagnostic probe to distinguish ordered from molten proteins *(*Bailey *et al., 2001)*. This structural difference was also observed between the HlyA and proHlyA *K*d values, demonstrating by an independent means that the fatty acids on the former induce a molten structure. Moreover, the higher fluorescence-transfer efficiency for HlyA compared to that for proHlyA indicated that the quenching of tryptophan fluorescence was more effective when the binding of ANS to the molten-globule conformation took place, where the accessibility of both the surface and inner tryptophan residues was increased. Thus, the capability of ANS to quench tryptophan fluorescence was seen to be correlated with the ANS-binding behavior.

Proteins with molten-globule-like regions are included in the category of intrinsically disordered proteins, as recently reviewed elsewhere (Dunker *et al*., 2001). Most of the disordered regions of proHlyA that were predicted through the use of the predictor of naturally disordered regions (PONDR) were located in the C-terminal half of the protein (Fig. 2). These domains could be related to the different steps in this toxin's mechanism of action from its export from the bacterium to pore formation in the target cell.

HlyA carries a carboxy-terminal–secretion signal located within the last 50–60 amino acids (Jarchau *et al.,* 1994). This region is predicted to be disordered; and although export of the toxin has been observed to be acylation-independent (Ludwig *et al.*, 1987), as mentioned above, the yield from extracellular transport for proHlyA was lower than that for HlyA. Consequently, covalently bound acyl chains can expose these signal regions and thus facilitate transport.

Intrinsically unstructured proteins can bind in several different patterns through a process termed *binding promiscuity*. The intrinsic lack of structure can confer functional advantages, including the ability to bind—perhaps in various conformations—to several different target cells. This binding promiscuity would furthermore explain the previously mentioned ambiguity in experimental determinations of the presence of a specific receptor for HlyA published to date (Lally *et al*., 1997), (Cortajarena *et al.*, 2001), (Valeva *et al.*, 2005).

Many studies have searched for the presence of a receptor for HlyA in different target cells. For example, CD11a and CD18, the two subunits of β2-integrin, were identified as cellsurface receptors that mediate HlyA toxicity in the human target cells HL60 (Lally *et al.*, 1997). This receptor was found in most circulating leukocytes (lymphocytes, neutrophils, monocytes, and macrophages). Despite the absence of studies identifying the protein region responsible for the interaction with this receptor, studies on the adenylate-cyclasecontaining hemolysin of *Bordetella pertusis* (CyaA)—another RTX toxin—revealed that the main integrin-interacting domain of CyaA is located in its glycine/aspartate-rich repeat region; which stretch is characteristic of all protein members of this family. These results allowed the identification of region 1166–1287 as a major CD11b-binding motif (Azami-El-Idrissi *et al.*, 2003). Because this domain is involved in calcium binding, the authors proposed that CyaA shifts from a disordered structure to an R-helical conformation upon calcium binding to the RTX motifs (Rose *et al.,* 1995); therefore, the speculation that the calcium-binding domain composed of glycine-rich tandem repeats corresponding to amino acids 550–850 of HlyA might be involved in the binding to β2-integrin is tempting. That these regions also match the disordered regions predicted and that acyl chains might be implicated in the exposure since the calcium-binding capacity of proHlyA is lower than that of HlyA, should also be borne in mind (Soloaga *et al.*, 1996).

As cited above in **Section 2.4**, another protein identified as a receptor of HlyA in horse erythrocytes is the glycoprotein glycophorin (Cortajarena *et al*., 2001). A glycophorinbinding region between residues 914 and 936 accordingly has been identified (Cortajarena *et al*., 2003). Previous sequence analyses of several RTX toxins had revealed that this stretch was a conserved region. If this region was deleted, the specific binding of HlyA to the cell-surface receptors on erythrocytes was lost without affecting its nonspecific binding (adsorption) to lipid bilayers. This region was also predicted to be intrinsically disordered.

Proteins with molten-globule-like regions are included in the category of intrinsically disordered proteins, as recently reviewed elsewhere (Dunker *et al*., 2001). Most of the disordered regions of proHlyA that were predicted through the use of the predictor of naturally disordered regions (PONDR) were located in the C-terminal half of the protein (Fig. 2). These domains could be related to the different steps in this toxin's mechanism of

HlyA carries a carboxy-terminal–secretion signal located within the last 50–60 amino acids (Jarchau *et al.,* 1994). This region is predicted to be disordered; and although export of the toxin has been observed to be acylation-independent (Ludwig *et al.*, 1987), as mentioned above, the yield from extracellular transport for proHlyA was lower than that for HlyA. Consequently, covalently bound acyl chains can expose these signal regions and thus

Intrinsically unstructured proteins can bind in several different patterns through a process termed *binding promiscuity*. The intrinsic lack of structure can confer functional advantages, including the ability to bind—perhaps in various conformations—to several different target cells. This binding promiscuity would furthermore explain the previously mentioned ambiguity in experimental determinations of the presence of a specific receptor for HlyA

Many studies have searched for the presence of a receptor for HlyA in different target cells. For example, CD11a and CD18, the two subunits of β2-integrin, were identified as cellsurface receptors that mediate HlyA toxicity in the human target cells HL60 (Lally *et al.*, 1997). This receptor was found in most circulating leukocytes (lymphocytes, neutrophils, monocytes, and macrophages). Despite the absence of studies identifying the protein region responsible for the interaction with this receptor, studies on the adenylate-cyclasecontaining hemolysin of *Bordetella pertusis* (CyaA)—another RTX toxin—revealed that the main integrin-interacting domain of CyaA is located in its glycine/aspartate-rich repeat region; which stretch is characteristic of all protein members of this family. These results allowed the identification of region 1166–1287 as a major CD11b-binding motif (Azami-El-Idrissi *et al.*, 2003). Because this domain is involved in calcium binding, the authors proposed that CyaA shifts from a disordered structure to an R-helical conformation upon calcium binding to the RTX motifs (Rose *et al.,* 1995); therefore, the speculation that the calcium-binding domain composed of glycine-rich tandem repeats corresponding to amino acids 550–850 of HlyA might be involved in the binding to β2-integrin is tempting. That these regions also match the disordered regions predicted and that acyl chains might be implicated in the exposure since the calcium-binding capacity of proHlyA is lower than that

As cited above in **Section 2.4**, another protein identified as a receptor of HlyA in horse erythrocytes is the glycoprotein glycophorin (Cortajarena *et al*., 2001). A glycophorinbinding region between residues 914 and 936 accordingly has been identified (Cortajarena *et al*., 2003). Previous sequence analyses of several RTX toxins had revealed that this stretch was a conserved region. If this region was deleted, the specific binding of HlyA to the cell-surface receptors on erythrocytes was lost without affecting its nonspecific binding (adsorption) to lipid bilayers. This region was also predicted to be intrinsically

published to date (Lally *et al*., 1997), (Cortajarena *et al.*, 2001), (Valeva *et al.*, 2005).

of HlyA, should also be borne in mind (Soloaga *et al.*, 1996).

action from its export from the bacterium to pore formation in the target cell.

facilitate transport.

disordered.

The role of fatty acids in the exposure of disordered regions is supported by results published for the D12-monoclonal-antibody–epitope reactivity. The D12 epitope maps to amino acids 673–726. Since the D12 monoclonal antibody reacts with HlyA, but not with proHlyA; the acylation of the former is directly responsible for the exposure of the epitope within this region (Pellett *et al.,* 1990), (Rowe *et al.*, 1994).

Fig. 2. ProHlyA is activated by acylation.

This process is catalyzed by HlyC, which species transfers a fatty acid from ACP to two internal lysines of ProHlyA (Lys 564 and Lys 690). Once covalently bound, these fatty acids induce a molten-globule conformation in HlyA that exposes intrinsically disordered regions, the existence of which zones was predicted by the predictor of naturally disordered regions PONDR. The amino-acid sequence is represented on the *x*-axis, and the prediction of disorder on the *y*-axis. Peaks >0.5 are strongly predicted to be disordered (Dunker *et al.,* 2005).

#### **3.2 Promotion of protein oligomerization**

Lipid binding to proteins can also be a determinant of specific protein-protein interactions such as the assembly of proteins into oligomeric complexes. This circumstance obtains for HlyA, where an oligomer was found at lytic concentrations in sheep-erythrocyte ghosts. In contrast, no oligomeric structure was found for proHlyA (Herlax *et al.*, 2009).

Fluorescence-Resonance-Energy Transfer (FRET) is a photochemical process whereby one fluorescent molecule or fluorophore, the "donor", upon excitation by an initial photon of light, spontaneously transfers its energy to another molecule, the "acceptor", by a nonradioactive dipole-dipole interaction (Forster, 1959). The distance over which energy can be transferred depends on the spectral characteristics of the fluorophores, but is generally within the 10–100-Å range. Hence, FRET can be used for measuring structure (Lakowicz *et al.*, 1990), conformational changes (Heyduk, 2002), and interactions between molecules (Parsons *et al.,* 2004). Since HlyA does not contain cysteine residues in its sequences, lysine 344 was replaced by a cysteine (HlyA K344C) and the same point mutation introduced into the unacylated protein (proHlyA K344C). The aim of this point mutagenesis was to permit the binding of only one fluorescent probe per protein, where that mutation—hopefully located in the insertion region of the toxin into membranes (Hyland *et al.*, 2001)—would not affect the hemolytic activity of the toxin. To carry out this study, two populations of HlyA K344C mutant proteins, one labelled with donor (Alexa-488) and the other with acceptor fluorophores (Alexa-546), were bound to sheep-erythrocyte ghosts. Our report showed that an oligomer was involved in the hemolytic mechanism of HlyA (Herlax *et al*., 2009). FRET can be used to study the distribution of molecules in membranes because the average spacing between molecules of interest will depend primarily on their lateral arrangement. Molecules may be within FRET distance either because they are clustered or because they are randomly distributed at such high surface densities that a fraction of them is within FRET proximity. The latter possibility was avoided in our experiments by using a high lipid/protein molar ratio (109) to insure that the observed FRET corresponded to oligomerization of the toxin on the erythrocyte surface. In comparison, the absence of FRET in the mutant protein, proHlyA K344C confirmed the participation of the covalently bound fatty acids in the oligomerization process. Fig. 3 shows the fluorescence spectra obtained in the FRET experiments for both proteins. *Prima facie*, this absence of FRET could be attributed to a reduced binding of the mutant protein to the erythrocyte ghosts, but this possibility was discarded because the percentage of binding to the membranes of both proteins was similar. We need to underscore here that fatty acids are essential for hemolytic activity; and considering that they are needed for oligomerization, we can state that oligomerization is necessary for hemolysis. We thus feel tempted to propose that the presence of fatty acids covalently bound to the protein leads to the exposure of regions that are implicated in protein-protein interactions.

In addition, a critical role of acylation in the oligomerization process to form hemolytic pores has been proposed for the adenylate-cyclase toxin from *Bordetella pertussis* (*cf*. **Section 3.1**) (Hackett *et al.*, 1995).

Finally, if we consider that pores formed by HlyA are sensitive to proteases on the *cis* side of the planar lipid membranes (Menestrina *et al.*, 1987), we could propose the possibility that the part of the toxin remaining external to the membrane is involved in the protein-protein interaction responsible for oligomerization and thus participates in pore formation.

Lipid binding to proteins can also be a determinant of specific protein-protein interactions such as the assembly of proteins into oligomeric complexes. This circumstance obtains for HlyA, where an oligomer was found at lytic concentrations in sheep-erythrocyte ghosts. In

Fluorescence-Resonance-Energy Transfer (FRET) is a photochemical process whereby one fluorescent molecule or fluorophore, the "donor", upon excitation by an initial photon of light, spontaneously transfers its energy to another molecule, the "acceptor", by a nonradioactive dipole-dipole interaction (Forster, 1959). The distance over which energy can be transferred depends on the spectral characteristics of the fluorophores, but is generally within the 10–100-Å range. Hence, FRET can be used for measuring structure (Lakowicz *et al.*, 1990), conformational changes (Heyduk, 2002), and interactions between molecules (Parsons *et al.,* 2004). Since HlyA does not contain cysteine residues in its sequences, lysine 344 was replaced by a cysteine (HlyA K344C) and the same point mutation introduced into the unacylated protein (proHlyA K344C). The aim of this point mutagenesis was to permit the binding of only one fluorescent probe per protein, where that mutation—hopefully located in the insertion region of the toxin into membranes (Hyland *et al.*, 2001)—would not affect the hemolytic activity of the toxin. To carry out this study, two populations of HlyA K344C mutant proteins, one labelled with donor (Alexa-488) and the other with acceptor fluorophores (Alexa-546), were bound to sheep-erythrocyte ghosts. Our report showed that an oligomer was involved in the hemolytic mechanism of HlyA (Herlax *et al*., 2009). FRET can be used to study the distribution of molecules in membranes because the average spacing between molecules of interest will depend primarily on their lateral arrangement. Molecules may be within FRET distance either because they are clustered or because they are randomly distributed at such high surface densities that a fraction of them is within FRET proximity. The latter possibility was avoided in our experiments by using a high lipid/protein molar ratio (109) to insure that the observed FRET corresponded to oligomerization of the toxin on the erythrocyte surface. In comparison, the absence of FRET in the mutant protein, proHlyA K344C confirmed the participation of the covalently bound fatty acids in the oligomerization process. Fig. 3 shows the fluorescence spectra obtained in the FRET experiments for both proteins. *Prima facie*, this absence of FRET could be attributed to a reduced binding of the mutant protein to the erythrocyte ghosts, but this possibility was discarded because the percentage of binding to the membranes of both proteins was similar. We need to underscore here that fatty acids are essential for hemolytic activity; and considering that they are needed for oligomerization, we can state that oligomerization is necessary for hemolysis. We thus feel tempted to propose that the presence of fatty acids covalently bound to the protein leads to the exposure of regions that are implicated in

In addition, a critical role of acylation in the oligomerization process to form hemolytic pores has been proposed for the adenylate-cyclase toxin from *Bordetella pertussis* (*cf*. **Section** 

Finally, if we consider that pores formed by HlyA are sensitive to proteases on the *cis* side of the planar lipid membranes (Menestrina *et al.*, 1987), we could propose the possibility that the part of the toxin remaining external to the membrane is involved in the protein-protein

interaction responsible for oligomerization and thus participates in pore formation.

contrast, no oligomeric structure was found for proHlyA (Herlax *et al.*, 2009).

**3.2 Promotion of protein oligomerization** 

protein-protein interactions.

**3.1**) (Hackett *et al.*, 1995).

Fig. 3. Oligomerization of HlyA in erythrocyte ghosts. FRET calculated for HlyA K344C and proHlyA K344C bound to erythrocyte ghosts. The lipid/protein ratio was 109. *Left inset*: Example of spectra measured for HlyA K344C. Fluorescence-emission spectrum of erythrocyte ghosts containing donor/acceptor, *F*D/A(480,λem) (excited at 480 nm; *blue triangle*), emission spectrum of erythrocyte ghosts labelled only with donor, *F*D(480, λem) (*violet circle*), emission spectrum of erythrocyte ghosts containing D/A, *F*D/A(530, λem) (excited at 530 nm where only the acceptor absorbs; *light blue square*), and emission spectrum of erythrocyte ghosts labelled only with acceptor, *F*A(480, λem) (*purple square*). *Right inset*: The same emission spectrum as in the left inset but measured for ProHlyA K344C.

#### **3.3 Contrary to expectations, fatty acids do not facilitate the interaction of HlyA with membrane microdomains**

A variety of pathogens and toxins have been recognized as interacting with microdomains in the plasma membrane. These microdomains are enriched in cholesterol and sphingolipids and probably exist in a liquid-ordered phase, in which lipid acyl chains are extended and ordered (Brown & London, 1998). Many proteins are targeted to these membrane microdomains by their favorable association with ordered lipids. Interestingly, such proteins are linked to saturated acyl chains, which species partition well into those domains (Pike, 2003). Although covalently bound fatty acids had not been implicated in the targeting of HlyA to membranes, their involvement in the targeting to membrane microdomains was studied (Herlax *et al*., 2009). For this purpose—and taking into account that these microdomains are enriched in cholesterol and sphingolipids—the hemolytic activity of the toxin on sheep erythrocytes was compared with the activity on cholesterol-depleted erythrocytes. The hemolysis rate of the cholesterol-poor erythrocytes was lower than that of the control erythrocytes at each HlyA concentration tested, thus pointing to the participation of cholesterol-enriched microdomains in the oligomerization process. For cholesterol-depleted erythrocytes, at low toxin concentrations, the kinetics of hemolysis seemed to be more complex, suggesting that toxin diffusion in membranes is the ratelimiting step. In order to determine if the decrease in the hemolytic rate observed in the cholesterol-depleted erythrocytes was caused by an impairment of toxin oligomerization, we repeated the FRET experiments comparing control and cholesterol-depleted sheeperythrocyte ghosts. We demonstrated that cholesterol depletion led to a decrease in FRET of 75% compared to the control sheep ghosts. This result indicated that cholesterolenriched microdomains played a significant role in the oligomerization process. To obtain more information about the effect of cholesterol-enriched microdomains within the oligomerization process, we performed FRET-kinetics experiments. The role of cholesterol was confirmed by the results of FRET kinetics, where the biphasic behavior of FRET suggested the initial formation of small oligomers, followed by their assembly to form multimeric structures (Fig. 4). The concentration of the small oligomers was favored by the cholesterol-enriched microdomains, where the diffusion time in the membrane became diminished. The number of HlyA molecules that became associated to form the

Fig. 4. *FRET kinetics.* Measurement of acceptor fluorescence at 570 nm as a function of time in a mixture composed of HlyA K344C labelled with fluorescent donor and acceptor plus either control erythrocytes (black line) or cholesterol-depleted erythrocytes (light gray line). Measurement of a mixture of unlabelled and labelled with acceptor HlyA K344C with control erythrocytes (*dark gray line*) was done as FRET-negative control. Assays were performed at a ratio of 5 µg of total toxin per 100 µg of phospholipids (as erythrocyte membranes). The excitation and emission monochromators were set at 480 nm and 570 nm, respectively. Alexa-546 emission was measured at a rate of 25 samples/s for 240 s, at 37°C. The *curves* represent the average value of three independent experiments containing five replicates each.

erythrocytes. The hemolysis rate of the cholesterol-poor erythrocytes was lower than that of the control erythrocytes at each HlyA concentration tested, thus pointing to the participation of cholesterol-enriched microdomains in the oligomerization process. For cholesterol-depleted erythrocytes, at low toxin concentrations, the kinetics of hemolysis seemed to be more complex, suggesting that toxin diffusion in membranes is the ratelimiting step. In order to determine if the decrease in the hemolytic rate observed in the cholesterol-depleted erythrocytes was caused by an impairment of toxin oligomerization, we repeated the FRET experiments comparing control and cholesterol-depleted sheeperythrocyte ghosts. We demonstrated that cholesterol depletion led to a decrease in FRET of 75% compared to the control sheep ghosts. This result indicated that cholesterolenriched microdomains played a significant role in the oligomerization process. To obtain more information about the effect of cholesterol-enriched microdomains within the oligomerization process, we performed FRET-kinetics experiments. The role of cholesterol was confirmed by the results of FRET kinetics, where the biphasic behavior of FRET suggested the initial formation of small oligomers, followed by their assembly to form multimeric structures (Fig. 4). The concentration of the small oligomers was favored by the cholesterol-enriched microdomains, where the diffusion time in the membrane became diminished. The number of HlyA molecules that became associated to form the

Fig. 4. *FRET kinetics.* Measurement of acceptor fluorescence at 570 nm as a function of time in a mixture composed of HlyA K344C labelled with fluorescent donor and acceptor plus either control erythrocytes (black line) or cholesterol-depleted erythrocytes (light gray line).

Measurement of a mixture of unlabelled and labelled with acceptor HlyA K344C with control erythrocytes (*dark gray line*) was done as FRET-negative control. Assays were performed at a ratio of 5 µg of total toxin per 100 µg of phospholipids (as erythrocyte membranes). The excitation and emission monochromators were set at 480 nm and 570 nm, respectively. Alexa-546 emission was measured at a rate of 25 samples/s for 240 s, at 37°C. The *curves* represent

the average value of three independent experiments containing five replicates each.

pore was uncertain; nevertheless, the assumption that several molecules could oligomerize to form a pore was not unreasonable. An extension of this reasoning suggested that at high doses a progressive oligomerization of HlyA leads to the fusion of the pore and rapid destruction of the cell membrane with little time for activation of the central apoptotic pathway. By contrast, at lower concentrations, the pores would be smaller and fewer in number so that the cells, though injured, would survive long enough for apoptosis to be observed (Lally *et al.*, 1997). These results can explain why toxin association with erythrocytes at 0–2°C is characterized as a prelytic state, whereas following a shift to 23°C—and after a lag period—lysis begins (Moayeri & Welch, 1997). In conclusion, the fusion of oligomers may be the rate-limiting step in pore formation, and the integrity of the cholesterol-enriched microdomains is necessary for the concentration of HlyA to induce hemolysis. This notion agrees with the findings of Moayeri and Welch (Moayeri & Welch, 1994), who observed that the degree of osmotic protection of erythrocytes afforded by protectants of varying sizes depended on the amount of toxin applied and the duration of the assay. These authors suggested that HlyA creates a lesion with a very small initial size that then increases in apparent diameter over time. Consequently, the larger the oligomer is, the bigger the pore size becomes.

That the terms "membrane microdomains" and "detergent-resistant microdomains" (DRMs) are not synonymous is essential to remember because the two have different origins and conceptual meanings (Lichtenberg *et al.*, 2005). The DRM technique, though, is widely used in the current literature to investigate the interaction between a protein and membrane microdomains. This technique takes advantage of the selective solubilization of different lipids that occurs when a biomembrane is submitted to the action of a nonionic detergent such as Triton X-100. When erythrocyte ghosts were incubated with HlyA and the DRMs were separated by sucrose-gradient ultracentrifugation, the immunoblot analysis revealed that most of the ghost-associated HlyA was localized in the DRMs, indicating that the binding of HlyA to the erythrocyte membranes was mediated by membrane microdomains that served as concentration platforms for the toxin's oligomerization. That proHlyA colocalizes with HlyA and flotillin (a microdomain protein marker) in DRMs emphasizes our hypothesis that the main role of the saturated acyl chain covalently bound to HlyA is a participation in the oligomerization process, and not the targeting to cholesterol-enriched membranes (Fig 5).

A key feature of cholesterol-enriched microdomains is the tight packing of lipid acyl chains in the liquid-ordered phase, where the lipid acyl chains are extended and ordered (Brown & London, 1998). Because of the difficulty in packing membrane-spanning helices into the ordered lipid environment, some proteins are linked to saturated acyl chains and partition well into those microdomains (de Planque & Killian, 2003). Shogomori *et al.* (2005) found, however, that acylation did not measurably enhance microdomain association, and they concluded that the acylated linker for the activation of T-cell transmembrane domains had a low inherent affinity for cholesterol-enriched microdomains. The possible inferrence is that acylation is not sufficient for the targeting of any transmembrane protein and that therefore a second mechanism—such as protein-protein interactions for microdomain associations—is required (Fragoso *et al.,* 2003), (Cherukuri *et al.*, 2004).

To conclude, we propose that fatty acids covalently bound to HlyA and membrane microdomains are implicated in the hemolysis process. Fatty acids are essential because they induce the exposure of intrinsic disordered regions in the toxin so as to enhance proteinprotein interactions in order to form the oligomer, while the membrane microdomains act as platforms for the concentration of the toxin during the oligomerization process.

Fig. 5. *Interaction of HlyA with DRMs.*Thirty µg of HlyA were incubated with 100 µl of erythrocyte ghosts for 30 min at 37 °C. Cells were lysed with 1% (v/v) Triton X-100 and insoluble cell components separated by sucrose-density-gradient centrifugation. The gradient fractions were analyzed by immunoblotting with anti-HlyA antibodies. HlyA was present in fractions 3 and 4. Gradient fractions were also analyzed by immunoblotting with anti-Flotillin-1 antibodies. Flotillin-1 appears mainly in fractions 3 and 4. ProHlyA was incubated under the same conditions as HlyA. The gradient fractions were analyzed by immunoblotting with anti-HlyA antibodies. ProHlyA colocalizes with HlyA.

In summary, fatty acids covalently bound to HlyA induce a molten-globule structure in the toxin, exposing intrinsically disordered regions involved in the different steps in the toxin's mechanism of action. Fatty acids expose specific regions that induce protein-protein interaction in the oligomerization process that takes place within the membrane microdomains of erythrocytes. The irreversibility of the toxin's membrane binding promoted by fatty acids might result from the formation of the HlyA oligomeric structure (Herlax & Bakas, 2003,2007), (Herlax *et al.*, 2009).

#### **4. Toxin-based theraphy**

Bacterial toxins have been defined as "soluble substances that alter the normal metabolism of host cells with deleterious effects on the host" (Schlessinger & Schaechter, 1993). Nonetheless, during the last decade, taking advantage of advances in toxin research, investigators have sought ways of obtaining benefits from toxins. In the present section we will discuss these toxin-based therapies and the possible relevant use of HlyA.

#### **4.1 Immunotoxins**

122 Biochemistry

induce the exposure of intrinsic disordered regions in the toxin so as to enhance proteinprotein interactions in order to form the oligomer, while the membrane microdomains act as

**HlyA** 

**Flotilin** 

**ProHlyA** 

platforms for the concentration of the toxin during the oligomerization process.

Fig. 5. *Interaction of HlyA with DRMs.*Thirty µg of HlyA were incubated with 100 µl of erythrocyte ghosts for 30 min at 37 °C. Cells were lysed with 1% (v/v) Triton X-100 and insoluble cell components separated by sucrose-density-gradient centrifugation. The gradient fractions were analyzed by immunoblotting with anti-HlyA antibodies. HlyA was present in fractions 3 and 4. Gradient fractions were also analyzed by immunoblotting with anti-Flotillin-1 antibodies. Flotillin-1 appears mainly in fractions 3 and 4. ProHlyA was incubated under the same conditions as HlyA. The gradient fractions were analyzed by

In summary, fatty acids covalently bound to HlyA induce a molten-globule structure in the toxin, exposing intrinsically disordered regions involved in the different steps in the toxin's mechanism of action. Fatty acids expose specific regions that induce protein-protein interaction in the oligomerization process that takes place within the membrane microdomains of erythrocytes. The irreversibility of the toxin's membrane binding promoted by fatty acids might result from the formation of the HlyA oligomeric structure

Bacterial toxins have been defined as "soluble substances that alter the normal metabolism of host cells with deleterious effects on the host" (Schlessinger & Schaechter, 1993). Nonetheless, during the last decade, taking advantage of advances in toxin research,

immunoblotting with anti-HlyA antibodies. ProHlyA colocalizes with HlyA.

(Herlax & Bakas, 2003,2007), (Herlax *et al.*, 2009).

**4. Toxin-based theraphy** 

Conventional cancer treatments such as surgery, chemotherapy, and radiotherapy often fail to achieve complete cancer remission. Moreover, radiotherapy and/or chemotherapy are almost always cause significant—and sometimes long-lasting—side effects. These considerations have prompted the development of many new approaches for the treatment of cancer. One such example involves the use of immunotoxins (Bernardes *et al*., 2010).

The term "immunotoxin" classically refers to chimeric proteins with a cell-selective ligand chemically linked or genetically fused to a toxin moiety that can target cancer cells overexpressing tumor-associated antigens, membrane receptors, or carbohydrate antigens. In the 1970s the first therapeutic agents composed of toxins conjugated to antibodies against cell-surface antigens started to emerge as tumor-cell killers (Moolten & Cooperband, 1970), (Moolten *et al.*, 1976). Since then, many hybrid molecules consisting of a toxin coupled to a specific targeting antibody or ligand were developed, with most of these hybrids being directed against tumor cells (Pastan *et al.*, 2007).

First-generation immunotoxins were prepared by chemically conjugating antibodies to natural intact toxin units or to toxins with attenuated cell-binding capability. These constructs, however, were heterogeneous and nonspecific both because multiplicities of potential sites were available for chemical conjugation and since the presence of the cellbinding domain on the toxin led to an intoxication of nontumor cells as well. Immunotoxins of the second generation were also based on chemical conjugation between the targeting moiety and the toxin. Nevertheless, accumulated knowledge on the structure and function of the toxins enabled the removal of their native nonspecific cell-binding domain, thus generating immunotoxins that were much more target-specific when conjugated to monoclonal antibodies. Although more specific and thus better tolerated by animals, immunotoxins from this second generation were still chemically heterogeneous, and their large size hindered them from penetrating solid tumors. In order to avoid this heterogeneity, improve tumor penetration, and reduce production complexity and expense, recombinant-DNA techniques were applied in the production of the third-generation immunotoxins. In these constructs—mostly produced in the bacterium *E. coli*—the cellbinding domain of the toxin is genetically replaced with a ligand or with the Fv portion of an antibody in which the immunoglobilin light- and heavy-chain variable regions are either genetically linked or held together by a disulfide bond (Shapira & Benhar, 2010).

Among the bacterial toxins that were used for the construction of immunotoxins, the most common were the diphtheria toxin and the pseudomonas exotoxin A, which toxins are naturally produced by the Gram-positive, aerobic *Corynebacterium diphtheria* and by the Gram-negative, aerobic *Pseudomonas aeruginosa*, respectively. Clinical trials with different exotoxin A-immunotoxins have already been performed with positive results in leukemia and bladder cancer (Kreitman *et al.,* 2001), (Kreitman *et al.,* 2005), (Biggers & Scheinfeld, 2008).

In spite of the promise shown by bacterial toxin-based chimeric proteins, these hybrids still present several obstacles that limit their clinical application. The toxin part of the fusion proteins elicits a high degree of humoral response in humans. In addition, in developed countries, where people have become immunized against diphtheria, the patient's serum will have circulating antibodies against the diphtheria toxin that will result in a neutralization of diphtheria toxin-based immunotoxins (Hall *et al.*, 2001). Both the *Pseudomonas* exotoxin and the diphtheria toxin are large molecules and are difficult to humanize. At sufficiently high concentrations these fusion proteins lead to symptoms like the vascular-leak syndrome and thus exhibit a certain degree of nonspecific toxicity.

#### **HlyA as a possible candidate toxin for the synthesis of immunotoxins**

Considering all the details of the structure and mode of action of HlyA discussed above, we can state that HlyA can be a good candidate for an effective immunotoxin. Although, certain additional details about the domains implicated in the binding of the toxin to target-cell receptors need to be clarified, we can consider that the domain that comprises amino acids 914–936 should be exchanged for the specific antibody (or ligand) chosen for interaction with the tumor cell of interest. Perhaps some amino acids within the repeat domain that might be involved in the interaction with the ß2-integrins should also be removed in the fusion protein.

#### **The reason for using HlyA in an immunotoxin**

An observation deserving emphasis is that the more relevant effects that HlyA produces during an infection are sublytic rather than cytolytic. Among these effects we must bear in mind the one related to the modulation of the host-signaling cascades, where HlyA was found to produce an inactivation of the PKB (*cf*. **Section 2.4**)—a key protein involved in several pathways related to host-cell survival, inflammatory responses, proliferation, and metabolism (Manning & Cantley, 2007), (Fayard *et al.,* 2005). By inactivating PKB, HlyA is able to fine-tune host responses related to the inflammatory- and apoptosis-signaling cascades that are initiated during the course of an infection. PKB inactivation is produced by an extracellular calcium-dependent, potassium-independent process requiring HlyA insertion into the host plasma membrane and subsequent pore formation. Calcium influx induces the activation of host-protein phosphatases that dephosphorylate PKB, inactivating it and finally inducing host-cell apoptosis. Thus, if a ligand directed at a specific tumor-cell receptor is fused with HlyA, that immunotoxin might induce the apoptosis of the desired cell.

The advantage of using HlyA is that the translocation of the immunotoxin into a tumor cell is not necessary, only its binding to the membrane where HlyA can insert itself and form the pore needed to execute its apoptotic action. Of course, these hypotheses are only possibilities that would warrant further investigation.

#### **4.2 Vaccines**

In recent years, an increase in the development of vaccination technology has taken place, but the ideal vaccine has not yet been found. In general terms, there are certain criteria that a vaccine must satisfy: it must be capable of eliciting the appropriate immune response; and it should be safe, stable, and reproducible (Perrie *et al*., 2008).

UTIs caused by UPEC still represent an enormous challenge for the development of vaccines targeted to induce an immunity that can either prevent the infectious agent from attaching

proteins elicits a high degree of humoral response in humans. In addition, in developed countries, where people have become immunized against diphtheria, the patient's serum will have circulating antibodies against the diphtheria toxin that will result in a neutralization of diphtheria toxin-based immunotoxins (Hall *et al.*, 2001). Both the *Pseudomonas* exotoxin and the diphtheria toxin are large molecules and are difficult to humanize. At sufficiently high concentrations these fusion proteins lead to symptoms like

Considering all the details of the structure and mode of action of HlyA discussed above, we can state that HlyA can be a good candidate for an effective immunotoxin. Although, certain additional details about the domains implicated in the binding of the toxin to target-cell receptors need to be clarified, we can consider that the domain that comprises amino acids 914–936 should be exchanged for the specific antibody (or ligand) chosen for interaction with the tumor cell of interest. Perhaps some amino acids within the repeat domain that might be involved in the interaction with the ß2-integrins should also be

An observation deserving emphasis is that the more relevant effects that HlyA produces during an infection are sublytic rather than cytolytic. Among these effects we must bear in mind the one related to the modulation of the host-signaling cascades, where HlyA was found to produce an inactivation of the PKB (*cf*. **Section 2.4**)—a key protein involved in several pathways related to host-cell survival, inflammatory responses, proliferation, and metabolism (Manning & Cantley, 2007), (Fayard *et al.,* 2005). By inactivating PKB, HlyA is able to fine-tune host responses related to the inflammatory- and apoptosis-signaling cascades that are initiated during the course of an infection. PKB inactivation is produced by an extracellular calcium-dependent, potassium-independent process requiring HlyA insertion into the host plasma membrane and subsequent pore formation. Calcium influx induces the activation of host-protein phosphatases that dephosphorylate PKB, inactivating it and finally inducing host-cell apoptosis. Thus, if a ligand directed at a specific tumor-cell receptor is fused with HlyA, that immunotoxin might induce the

The advantage of using HlyA is that the translocation of the immunotoxin into a tumor cell is not necessary, only its binding to the membrane where HlyA can insert itself and form the pore needed to execute its apoptotic action. Of course, these hypotheses are only

In recent years, an increase in the development of vaccination technology has taken place, but the ideal vaccine has not yet been found. In general terms, there are certain criteria that a vaccine must satisfy: it must be capable of eliciting the appropriate immune response; and it

UTIs caused by UPEC still represent an enormous challenge for the development of vaccines targeted to induce an immunity that can either prevent the infectious agent from attaching

the vascular-leak syndrome and thus exhibit a certain degree of nonspecific toxicity.

**HlyA as a possible candidate toxin for the synthesis of immunotoxins** 

removed in the fusion protein.

apoptosis of the desired cell.

**4.2 Vaccines** 

**The reason for using HlyA in an immunotoxin** 

possibilities that would warrant further investigation.

should be safe, stable, and reproducible (Perrie *et al*., 2008).

to and colonizing the mucosal epithelium and/or can block the binding and action of microbial toxins, such as HlyA (Holmgren *et al.*, 2003). Since the infections by these bacteria occur at, or take their departure from, a mucosal surface; a mucosal route of vaccination should be selected rather than a parenteral one.

A significant aspect of immune responses at mucosal surfaces is the production of a secretory IgA (S-IgA) and its transport across the epithelium. This S-IgA response represents the first line of defence against the invasion by bacterial pathogens. The mucosal immune system is an integrated network of tissues, cells, and effector molecules that functions to protect the host from those pathogens. Furthermore, mucosal lymphocytes exhibit unique homing receptors, the integrins, that recognize ligands expressed on mucosal endothelial cells so as to allow their retention within mucosal tissues for the delivery of cellular and humoral immune responses (Butcher & Picker, 1996). Because of the presence of specific interconnected mucosal induction and effector sites for eliciting the S-IgA antibody response, the mucosal immune system has been shown to be separated from the peripheral immune system. Thus, the induction of peripheral immune responses by parenteral immunization does not necessarily result in significant mucosal immunity; by contrast, mucosal immunization is capable of inducing immune protection in both the external secretions and the peripheral immune compartments (Kiyono *et al.*, 1992), (McGhee *et al.*, 1992).

The induction of immune responses following mucosal immunization is usually dependent upon the coadministration of the appropriate adjuvants that can initiate and support the transition from innate to adaptive immunity. While a number of substances of bacterial origin have been tested as mucosal adjuvants, the most widely used mucosal adjuvants in experimental animals are the cholera toxin (CT) and the closely related *E. coli* heat-labile enterotoxin (LT). Both CT and LT consist in homopentamers of cell-binding B subunits associated with a single toxically active A subunit. The A subunit enzymatically ADPribosylates the Gs protein of adenylate cyclase leading to an increased cAMP production in the affected cells (de Haan & Hirst, 2000). CT and LT can alter several steps in the induction of a mucosal immune response. These effects, which alone or in combination might explain their strong adjuvant action after oral immunization, include: (1) an increased permeability of the intestinal epithelium leading to an enhanced uptake of a coadministered antigen, (2) an enhanced antigen presentation by various antigen-presenting cells, (3) a promotion of isotype differentiation in B cells leading to increased IgA formation, and (4) complex stimulatory as well as inhibitory effects on T-cell proliferation and cytokine production. Finally, CT and LT have been shown not only to avoid an induction of oral tolerance but also to abrogate additional otherwise efficient regimens producing tolerance induction after oral antigen administration (Holmgren *et al.,* 2005).

A number of studies have examined the activity of LT and CT as mucosal adjuvants in vaccines against a variety of bacterial, fungal, and viral pathogens. Representative examples include the tetanus toxoid (Xu-Amano *et al.*, 1993), (Yamamoto *et al.*, 1996), (Cheng *et al.*, 1999), (Xu-Amano *et al.,* 1994), the inactivated influenza virus (Hashigucci *et al.,* 1996), (Tumpey *et al.*, 2001), a recombinant urease from *Helicobacter* spp. (Lee *et al.*, 1995), (Weltzin *et al.,* 1997), (Lee, 2001), and the pneumococcal surface protein A from *S. pneumoniae* (Wu *et al.*,1997). Many other examples have been reported, and all these studies clearly indicate that both LT and CT have significant potential for use as adjuvants for mucosally administered antigens. Nevertheless, both LT and CT are potent enterotoxins, and this property has seriously limited the practical use of these molecules (Freytag & Clements 2005). To avoid such toxicity, a number of nontoxic mutant derivatives of CT or LT have been proposed (Douce G, 1997) (Douce *et al*. 1998). In particular, single-amino-acid-substitution mutants of LT (R7K, S63K and R192G) that lack ADP-ribosyltransferase activity have been shown to retain their adjuvant properties (Douce et al. 1995).

In contrast, because of their size, plasticity, and safety profile in humans, OMVs are attractive vehicles for vaccine delivery. OMV vaccines for serogroup-B meningococcal disease, consisting in vesicles from *Salmonella thyphimurium* and *Pseudomonas aeruginosa* that contain surface antigens native to the pathogens have been shown to exhibit immunogenic properties (Alaniz *et al*., 2007), (Bauman & Kuehn, 2006). Two vaccines for serogroup-B meningococcal disease currently exist that are formulations comprising bacterial surface antigens that have been naturally incorporated into OMVs (Oster *et al*., 2005), (Feiring *et al*. , 2006). These OMVbased vaccines represent a novel system where both the antigen and delivery vehicle are derived from the *Neisseria meningitidis* pathogen itself (Claassen *et al*., 1996), (Arigita *et al.,* 2004). Moreover, genetically engineered OMVs offer an attractive possibility for use as easily purified vaccine-delivery systems capable of greatly enhancing the immunogenicity of lowimmunogenicity protein antigens without the need for an added adjuvant.

With the development of controlled-release technologies, the engineering of OMVs emerged as a promising strategy for antigen delivery because these vesicles are similar in geometry to naturally occurring pathogens and are readily internalized by antigen-presenting cells, thus avoiding the complex manufacturing steps required to purify and encapsulate antigens into particulate delivery systems such as polymer particles (Singh *et al.*, 2007), immunestimulating complexes (Morein *et al.,* 1984), liposomes, proteosomes, and related vesicles (Lowell *et al*., 1988), (Lowell *et al.*, 1988), (Felnerova *et al*., 2004), (Copland *et al.*, 2005)—all of which processes render these approaches economically unfeasible (Ulmer *et al.*, 2006).

The genetic fusion of the green-fluorescent protein (GFP) as a model subunit antigen with the bacterial hemolysin ClyA resulted in a chimeric protein that elicited strong anti-GFP antibody titers in immunized mice, whereas immunization with GFP alone elicited no such titers. Similar to native unfused ClyA, the chimeric ClyA-fusion proteins were found localized in bacterial OMVs, where they retained the activity of the fusion partners, thus demonstrating for the first time that ClyA can be used to colocalize fully functional heterologous proteins directly in bacterial OMVs. The anti-GFP humoral response in mice immunized with the engineered OMV formulations was indistinguishable from the response to the purified ClyA-GFP fusion protein alone and was equal to the response to purified proteins adsorbed to aluminum hydroxide, a standard adjuvant. Engineered OMVs containing ClyA-GFP were easily isolated by ultracentrifugation, thus effectively eliminating the need for a laborious antigen purification from cell-culture expression systems (Chena *et al*., 2010). The retention of hemolytic-protein activity indicated that ClyA-antigen fusions maintained their conformations. Although no pathologic effects were observed in mice immunized with ClyA, a detoxification of the toxin through mutation, truncation, or chemical methods may attenuate any possible toxicity while still retaining the hybrid's immunomodulatory capabilities.

On the basis of all these data, HlyA presents many properties that can be considered when designing a vaccine.

antigens. Nevertheless, both LT and CT are potent enterotoxins, and this property has seriously limited the practical use of these molecules (Freytag & Clements 2005). To avoid such toxicity, a number of nontoxic mutant derivatives of CT or LT have been proposed (Douce G, 1997) (Douce *et al*. 1998). In particular, single-amino-acid-substitution mutants of LT (R7K, S63K and R192G) that lack ADP-ribosyltransferase activity have been shown to

In contrast, because of their size, plasticity, and safety profile in humans, OMVs are attractive vehicles for vaccine delivery. OMV vaccines for serogroup-B meningococcal disease, consisting in vesicles from *Salmonella thyphimurium* and *Pseudomonas aeruginosa* that contain surface antigens native to the pathogens have been shown to exhibit immunogenic properties (Alaniz *et al*., 2007), (Bauman & Kuehn, 2006). Two vaccines for serogroup-B meningococcal disease currently exist that are formulations comprising bacterial surface antigens that have been naturally incorporated into OMVs (Oster *et al*., 2005), (Feiring *et al*. , 2006). These OMVbased vaccines represent a novel system where both the antigen and delivery vehicle are derived from the *Neisseria meningitidis* pathogen itself (Claassen *et al*., 1996), (Arigita *et al.,* 2004). Moreover, genetically engineered OMVs offer an attractive possibility for use as easily purified vaccine-delivery systems capable of greatly enhancing the immunogenicity of low-

With the development of controlled-release technologies, the engineering of OMVs emerged as a promising strategy for antigen delivery because these vesicles are similar in geometry to naturally occurring pathogens and are readily internalized by antigen-presenting cells, thus avoiding the complex manufacturing steps required to purify and encapsulate antigens into particulate delivery systems such as polymer particles (Singh *et al.*, 2007), immunestimulating complexes (Morein *et al.,* 1984), liposomes, proteosomes, and related vesicles (Lowell *et al*., 1988), (Lowell *et al.*, 1988), (Felnerova *et al*., 2004), (Copland *et al.*, 2005)—all of which processes render these approaches economically unfeasible (Ulmer *et al.*, 2006).

The genetic fusion of the green-fluorescent protein (GFP) as a model subunit antigen with the bacterial hemolysin ClyA resulted in a chimeric protein that elicited strong anti-GFP antibody titers in immunized mice, whereas immunization with GFP alone elicited no such titers. Similar to native unfused ClyA, the chimeric ClyA-fusion proteins were found localized in bacterial OMVs, where they retained the activity of the fusion partners, thus demonstrating for the first time that ClyA can be used to colocalize fully functional heterologous proteins directly in bacterial OMVs. The anti-GFP humoral response in mice immunized with the engineered OMV formulations was indistinguishable from the response to the purified ClyA-GFP fusion protein alone and was equal to the response to purified proteins adsorbed to aluminum hydroxide, a standard adjuvant. Engineered OMVs containing ClyA-GFP were easily isolated by ultracentrifugation, thus effectively eliminating the need for a laborious antigen purification from cell-culture expression systems (Chena *et al*., 2010). The retention of hemolytic-protein activity indicated that ClyA-antigen fusions maintained their conformations. Although no pathologic effects were observed in mice immunized with ClyA, a detoxification of the toxin through mutation, truncation, or chemical methods may attenuate

any possible toxicity while still retaining the hybrid's immunomodulatory capabilities.

designing a vaccine.

On the basis of all these data, HlyA presents many properties that can be considered when

immunogenicity protein antigens without the need for an added adjuvant.

retain their adjuvant properties (Douce et al. 1995).

**Anti- UPEC vaccine:** The urinary tract is one of the most common sites of bacterial infection. As mentioned above, over half (53%) of all women along with 14% of men experience at least one UTI in their lifetime (Griebling, 2005), (Griebling, 2005). *E. coli* is the infectious agent in more than 80% of the uncomplicated UTIs (Marrs *et al.*, 2005), (Foxman & Brown, 2003). In addition, the upper UTIs of young children can cause permanent kidney damage. An estimated 57% of children with acute pyelonephritis develop renal scarring (Lin *et al.,* 2003).

In recent years, an increase in the antibiotic resistance of UPEC isolates has been observed (Bours *et al.*, 2010) that imposed an urgent need for alternative treatment and prevention strategies to combat this serious and widespread human pathogen. With this aim, much research has been focussed on the development of vaccines to stimulate protective immunity against UPEC. In those studies, surface-exposed molecules such as P fimbriae, the lipopolysaccharide core, α-hemolysin, and the salmochelin receptor IroN have been utilized as antigens for subunit vaccines (Goluszko *et al.*, 2005), (Russo *et al.*,2003), (O'Hanley *et al.,* 1991); but the limited success of these strategies prevented any vaccine from being currently available. One consideration is that this vaccine has to generate immune responses at the level of mucosal surfaces.

Large-scale reverse-vaccinology approaches offer an alternative to the traditional vaccine design through applying genomic and bioinformatic methods to identify novel vaccine targets (Pizza *et al*., 2000). Using this technique, Alteri *et al* identified a class of molecules involved in iron acquisition as vaccine candidates and reported that intranasal immunization with this UPEC outer-membrane iron receptor generated an antigenspecific humoral response to provide protection from UTI (Alteri *et al.*, 2009). The authors proposed that the targeting of an entire class of molecules instead a single protein would permit the identification of components of a more generally protective UTI vaccine and that this strategy could be used in the development of vaccines to prevent infections caused by other pathogenic bacteria. During this present year, these same authors, using the *in-vivo*-induced–antigen technology, identified a novel UPEC virulence factor (*tosA*, a gene encoding a predicted repeat-in-toxin family member) that could be useful as a potential vaccine target (Vigil *et al*., 2011). Although this methodology did not identify HlyA as a potential candidate for this vaccine, the introduction of that toxin would be beneficial. First of all, HlyA has been recognized as one of the main virulence factors associated with the pathogenicity caused by UPEC (Wiles, Kulesus and Mulvey 2008); second, the toxin induced an immunity response in host organisms (O'Hanley *et al*., 1991) and thus is immunogenic in its native state; third, it can also produce focal leaks in intestinal epithelia (Troeger *et al.,* 2007). Focal leaks are small openings within the epithelium where bacterial penetration occurs. HlyA induces such focal leaks in a proinflammatory environment—those being also induced by the secretion of the cytokines TNFα and IL-13. Of relevance to highlight is that HlyA can increase the permeability of the intestinal epithelium so as to lead to an enhanced uptake of a coadministered antigen, thus acting as both a coadjuvant and an antigen in its own right. The dose that induces this effect would naturally have to be extensively investigated.

HlyA can also be used as adjuvants in any other vaccine design against another pathogen. For example, the toxin can be included in any liposomal vaccine in order to facilitate uptake though epithelia for the induction of immunity.

**OMV vaccines**: Balsalobre *et al* (2006) demonstrated that physiologically active HlyA is associated with the OMVs produced from *E. coli* laboratory strains and also from natural and clinical isolates. In our laboratory, we found that the unacylated toxin (proHlyA) can also be associated with OMVs (Herlax *et al.*, 2010). On the basis of this finding, OMV vaccines can be designed by effecting a fusion of the desired antigen with ProHlyA. In this way, ProHlyA would direct the exposure of the antigen on the surface of the OMVs without inducing any cytotoxic response. An advantage of OMV vaccines is that, because of their size and lipopolysaccharide content, they are able to induce an adequate immune response.

Finally, mention must be made that these hypotheses are just speculative on the basis of what is known about the structure and function of HlyA, whose application in toxin-based therapy still has to be exhaustively investigated and especially the immune response the toxin might evoke.
