**2. The alpha-hemolysin (HlyA) of** *E. coli*

Extraintestinal pathogenic *Escherichia coli* (ExPEC) is the causative agent of at least 80% of all uncomplicated urinary-tract infections (UTIs), which pathologies currently rank among the most common of infectious diseases worldwide (Marrs et al. 2005), (Foxman & Brown, 2003). ExPEC strains that cause a UTI are called uropathogenic *E. coli* (UPEC). This unique group of *E. coli* strains can reside in the lower gastrointestinal tract of healthy adults (Foxman *et al.*, 2002), (Yamamoto *et al.,* 1997), but upon entry into the urinary tract can ascend to and colonize the bladder, causing cystitis. The infection may be confined to the bladder, or bacteria may ascend into the ureters to infect the kidneys and cause pyelonephritis. In severe cases, bacteria can further disseminate across the proximaltubular and capillary endothelia to the bloodstream, causing bacteremia (Mobley *et al.*, 2009.). A significant proportion of UTIs occur in patients with no known abnormalities of the urinary tract—the so-called *uncomplicated UTIs*. Certain host characteristics, however, such as a congenital defect in urinary-tract anatomy, are considered complicating factors for UTI and accordingly increase susceptibility to this infection as well as affect its diagnosis and management (Foxman, 2002.). Finally, colonization of the bladder in high numbers may occur without eliciting symptoms in the host, a condition known as asymptomatic bacteriuria (Hooton *et al*., 2000.). In recent years, an enormous amount of information has accrued through sequencing the genomes of several ExPEC patients. These data, together with epidemiological analyses, have confirmed that different ExPEC pathotypes share many known as well as putative virulence factors. These latter include a number of secreted toxins, iron-acquisition systems, adhesins, and capsular antigens (Wiles *et al.*, 2008). Secreted toxins—which proteins include **HlyA**, the cytotoxic necrotizing factor-1 (CNF-1), and the secreted autotransporter—can alter host signaling cascades, disrupt inflammatory responses, and induce host-cell death; whereas bacterial siderophores like aerobactin, bacteriocin, and enterobactin allow the ExPEC to sequester iron away from the host (Guyer *et al.*, 2002), (Wiles *et al.*, 2008). Adhesive organelles can mediate ExPEC interaction with, and entry into, host cells and tissues; while the expression of encapsulation may enable ExPEC to more effectively avoid professional phagocytes (Wiles *et al.*, 2008), (Dhakal *et al*. 2008).

Experiments in murine and cell-culture model systems have demonstrated that high levels of HlyA can cause the osmotic lysis of host cells, while sublytic concentrations of this poreforming toxin can modulate host-survival pathways by interfering with phagocyte chemotaxis (Wiles *et al*, 2008),(Jonas *et al.*, 1993), (Cavalieri & Snyder, 1982), (Chen *et al.,* 2006). Both HlyA and CNF-1 may in addition stimulate the breakdown of tissue barriers and the release of nutrients (Smith *et al.* 2008), (Bauer & Welch, 1996), but through the use of the

introducing HlyA, its synthesis, posttranslational modification, secretion, and activity; this chapter will focus on the role that covalently bound fatty acids play in the toxin's

In recent decades, scientific advances have permitted the manipulation of toxins by using different strategies for directing toxic moieties to diseased cells and tissues. The end of the chapter will involve a discussion of this so-called *toxin-based therapy* and the potential use of

Extraintestinal pathogenic *Escherichia coli* (ExPEC) is the causative agent of at least 80% of all uncomplicated urinary-tract infections (UTIs), which pathologies currently rank among the most common of infectious diseases worldwide (Marrs et al. 2005), (Foxman & Brown, 2003). ExPEC strains that cause a UTI are called uropathogenic *E. coli* (UPEC). This unique group of *E. coli* strains can reside in the lower gastrointestinal tract of healthy adults (Foxman *et al.*, 2002), (Yamamoto *et al.,* 1997), but upon entry into the urinary tract can ascend to and colonize the bladder, causing cystitis. The infection may be confined to the bladder, or bacteria may ascend into the ureters to infect the kidneys and cause pyelonephritis. In severe cases, bacteria can further disseminate across the proximaltubular and capillary endothelia to the bloodstream, causing bacteremia (Mobley *et al.*, 2009.). A significant proportion of UTIs occur in patients with no known abnormalities of the urinary tract—the so-called *uncomplicated UTIs*. Certain host characteristics, however, such as a congenital defect in urinary-tract anatomy, are considered complicating factors for UTI and accordingly increase susceptibility to this infection as well as affect its diagnosis and management (Foxman, 2002.). Finally, colonization of the bladder in high numbers may occur without eliciting symptoms in the host, a condition known as asymptomatic bacteriuria (Hooton *et al*., 2000.). In recent years, an enormous amount of information has accrued through sequencing the genomes of several ExPEC patients. These data, together with epidemiological analyses, have confirmed that different ExPEC pathotypes share many known as well as putative virulence factors. These latter include a number of secreted toxins, iron-acquisition systems, adhesins, and capsular antigens (Wiles *et al.*, 2008). Secreted toxins—which proteins include **HlyA**, the cytotoxic necrotizing factor-1 (CNF-1), and the secreted autotransporter—can alter host signaling cascades, disrupt inflammatory responses, and induce host-cell death; whereas bacterial siderophores like aerobactin, bacteriocin, and enterobactin allow the ExPEC to sequester iron away from the host (Guyer *et al.*, 2002), (Wiles *et al.*, 2008). Adhesive organelles can mediate ExPEC interaction with, and entry into, host cells and tissues; while the expression of encapsulation may enable ExPEC to more effectively avoid professional

Experiments in murine and cell-culture model systems have demonstrated that high levels of HlyA can cause the osmotic lysis of host cells, while sublytic concentrations of this poreforming toxin can modulate host-survival pathways by interfering with phagocyte chemotaxis (Wiles *et al*, 2008),(Jonas *et al.*, 1993), (Cavalieri & Snyder, 1982), (Chen *et al.,* 2006). Both HlyA and CNF-1 may in addition stimulate the breakdown of tissue barriers and the release of nutrients (Smith *et al.* 2008), (Bauer & Welch, 1996), but through the use of the

mechanism of action.

HlyA in that modality.

**2. The alpha-hemolysin (HlyA) of** *E. coli* 

phagocytes (Wiles *et al.*, 2008), (Dhakal *et al*. 2008).

zebrafish infection model phagocytes were found that appeared to be the primary targets of these toxins (Wiles *et al.*,2009).

HlyA represents the prototype of the first RTX family of proteins characterized by Rodney Welch (Welch 1991)*.* Produced by a variety of Gram-negative bacteria, these proteins exhibit two common features: The first is the presence of arrays of glycine- and aspartate-rich nonapeptide repeats, which sequences are located at the C-terminal portion. The second is the unique mode of secretion via the type-I system (an ABC-binding–cassette transporter). This first group of RTX toxins consists of toxins—mostly exhibiting cytotoxic pore-forming activity—that often are first detected as a hemolytic halo surrounding bacterial colonies grown on blood-agar plates (Muller *et al.*, 1983), (Welch, 1991), *(Felmlee et al.*, 1985). Recently, a subgroup of very large RTX toxins (>3200 residues) were discovered with multiple activities, such as protease and lipase. These pathogens were named the multifunctional autoprocessing RTX toxins, with the *Vibrio cholerae* toxin being the prototype of this group. In summary, the RTX proteins form a large and diverse family with a broad spectrum of biological and biochemical activities (Linhartova, *et al*., 2010).
