**2.4 The mechanism of action of HlyA**

HlyA belongs to one class of a wide range of host-cell-specific toxins. HlyA acts on a variety of cell types from several species—*e. g.*, red blood cells, embryo and adult fibroblasts, granulocytes, lymphocytes, and macrophages (Cavalieri *et al.*, 1984)—and also binds to and disrupts protein-free liposomes (Ostolaza *et al.,* 1993).

The host environments encountered by the ExPEC are extremely nutrient-poor; and the function of HlyA has generally been thought to be primarily the destruction of host cells, thereby facilitating the release of nutrients and other factors, such as iron, that are critical for bacterial growth. The lytic mechanism of HlyA is a complex process. Three stages seem to be involved that ultimately lead to cell lysis: binding, insertion, and oligomerization of the toxin within the membrane.

Studies that have explored the binding of HlyA to membranes and the characterization of a putative toxin-specific receptor have produced contradictory results. First, the lymphocyte function-associated antigen (LFA-1) (CD11a/CD18; α1β2 integrin), was reported to serve as the receptor for HlyA on polymorphonuclear neutrophils (Lally *et al.,* 1997) and HlyA was found to recognize and bind the N-linked oligosaccharides to their β2-integrin receptors (Morova *et al.*, 2008). This finding raises the possibility that the initial binding of the toxin to various cells might occur through the recognition of glycosylated membrane components, such as glycoproteins and gangliosides. Recently, Cortajarena *et al.* (2001) found that HlyA binds to the glycophorin of horse erythrocytes and that this binding was abolished by a

Although HlyA has its own machinery for export from the bacteria, the presence of a physiologically active HlyA in the outer-membrane vesicles (OMVs) of clinical-hemolytic (Balsalobre *et al.*, 2006) as well as laboratory-recombinant strains of *E. coli* (Herlax *et al.,* 2010)

OMVs are constantly being discharged from the surface of Gram-negative bacteria during bacterial growth. All Gram-negative bacteria studied to date, including *E. coli*, produce OMVs; and their release is increased when the bacteria are exposed to stressful conditions such as antibiotics or serum. Even though the release of OMVs could not be demonstrated *in vivo*, the presence of particles resembling those vesicles has, in fact, been detected in plasma from patients with different infectious processes (Beveridge, 1999). OMVs serve as secretory vehicles for the proteins and lipids of Gram-negative bacteria and in this manner play roles in establishing a colonization niche for carrying or transmitting virulence factors into host cells or otherwise modulating the host defense and response, thus acting as well as longrange virulence factors that can protect luminal cargo from extracellular host proteases and so penetrate into tissues more readily than the larger bacteria (Kuehn & Kesty, 2005). In addition to toxin-protein delivery, other roles have been characterized for OMVs—namely, interspecies interaction and communication during multispecies infections plus DNA uptake and transfer (Mashburn-Warren & Whiteley, 2006). In the particular example of HlyA, we have demonstrated that the toxin secreted in this way is transferred to the target cell in a concentrated manner and as such is more hemolytically efficient than the free HlyA (Herlax *et al.*, 2010). Moreover, Balsalobre *et al.* (2006) demonstrated that the HlyA associated with OMVs is protected from the attack of proteases, thus facilitating the survival

HlyA belongs to one class of a wide range of host-cell-specific toxins. HlyA acts on a variety of cell types from several species—*e. g.*, red blood cells, embryo and adult fibroblasts, granulocytes, lymphocytes, and macrophages (Cavalieri *et al.*, 1984)—and also binds to and

The host environments encountered by the ExPEC are extremely nutrient-poor; and the function of HlyA has generally been thought to be primarily the destruction of host cells, thereby facilitating the release of nutrients and other factors, such as iron, that are critical for bacterial growth. The lytic mechanism of HlyA is a complex process. Three stages seem to be involved that ultimately lead to cell lysis: binding, insertion, and oligomerization of the

Studies that have explored the binding of HlyA to membranes and the characterization of a putative toxin-specific receptor have produced contradictory results. First, the lymphocyte function-associated antigen (LFA-1) (CD11a/CD18; α1β2 integrin), was reported to serve as the receptor for HlyA on polymorphonuclear neutrophils (Lally *et al.,* 1997) and HlyA was found to recognize and bind the N-linked oligosaccharides to their β2-integrin receptors (Morova *et al.*, 2008). This finding raises the possibility that the initial binding of the toxin to various cells might occur through the recognition of glycosylated membrane components, such as glycoproteins and gangliosides. Recently, Cortajarena *et al.* (2001) found that HlyA binds to the glycophorin of horse erythrocytes and that this binding was abolished by a

has recently been demonstrated.

of the toxin within the adverse medium of a patient's plasma.

disrupts protein-free liposomes (Ostolaza *et al.,* 1993).

**2.4 The mechanism of action of HlyA** 

toxin within the membrane.

trypsinization of the membranes. In addition, these authors found that the glycophorin purified from erythrocyte ghosts and reconstituted in liposomes significantly increased liposomal sensitivity to HlyA. Amino acids 914-936 of HlyA were subsequently hypothesized to be responsible for binding to the ghost receptor (Cortajarena *et al.*, 2003).

Other studies, however, indicated that the binding of HlyA to cells occurred in a nonsaturable manner and that the toxin did not interact with a specific protein receptor either on granulocytes or erythrocytes (Valeva *et al*., 2005). Nevertheless, HlyA produces protein-free liposome disruption. Ostolaza *et al.* have reported that HlyA causes the release of fluorescent solutes following a so-called *all-or-none* mechanism. Using large unilamellar vesicles of different lipid compositions, the authors found that the vesicles composed of phosphatidylcholine, phosphatidylethanolamine, and cholesterol at a molar ratio of 2:1:1 were the most sensitive (Ostolaza *et al.*, 1993). These results demonstrated that the presence of a receptor was not necessary for hemolysis to occur. These contradictory findings regarding the presence or absence of a toxin-specific receptor might be related to the different amounts of toxin and/or the different types and animal species of target cells used in the various studies. At all events, the interaction of HlyA with a target-cell membrane devoid of any specific proteinaceous receptor appears to occur in two steps: an initial reversible adsorption of the toxin that is sensitive to electrostatic forces followed by an irreversible membrane insertion (Bakás *et al.,* 1996), (Ostolaza *et al.,* 1997). Studies with the isolated calcium-binding domain of HlyA revealed that that part of the protein may be adsorbed onto the membrane during the early stages of HlyA-membrane interaction (Sanchez-Magraner *et al.*, 2007).

The next step in the hemolytic process is the insertion of the toxin into the membrane. Hyland *et al.* (2001) demonstrated that the major region of HlyA that inserts into the membrane is located between residues 177 and 411. The insertion is furthermore independent of membrane lysis since HlyA-protein mutants that are completely nonlytic can insert into lipid monolayers (Sanchez-Magraner *et al.*, 2006). In addition, a binding of calcium to the toxin was shown to induce a protein conformational change that made the insertion process irreversible (Sanchez-Magraner *et al.*, 2006), (Bakás *et al.* 1998). Once the toxin is inserted, an oligomerization process occurs. We previously found that the fatty acids covalently bound to the toxin induce conformational changes that expose intrinsically disordered regions so as to promote protein-protein interactions. Thus, the oligomerization process of the toxin is facilitated by microdomains within the membrane (Herlax & Bakas, 2007), (Herlax *et al.,* 2009).

The HlyA pore that is formed is highly dynamic because the size depends on both the interaction time and the concentration of the toxin (Welch, 2001). We recently demonstrated that the pore is of a proteolipidic nature since the conductance and membrane lifetime are dependent on membrane composition (Bakas *et al.,* 2006).

Nevertheless, what is not clear is how often HlyA reaches levels that are high enough to lyse host target cells during the course of an infection. In fact, sublytic concentrations of HlyA may even be more physiologically relevant. Indeed, recent studies have demonstrated that sublytic concentrations of a number of pore-forming toxins can modulate a variety of host signaling pathways, including the transient stimulation of calcium oscillations, the activation of MAP-kinase signaling, and the alteration of histone-phosphorylation and -

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