Escherichia coli O157H7

E. coli is one of the most well-studied bacterium in the microbiology field due to its frequent incidence in different environments and hosts, as well as its use as a tool in molecular biology. Currently, there are several categories of E. coli known to cause disease, mainly diarrhea in humans, also named as diarrheiogenic E. coli. Among those, enterohemorrhagic E. coli O157:H7 (EHEC) is one of the most important pathogenic E. coli. EHEC has been associated with several recent food-borne outbreaks of bloody diarrhea and hemolytic uremic syndrome (HUS) throughout the world. EHEC has an unusually low infectious dose when compared with other enteric bacterial pathogens such as Vibrio cholera and Salmonella enterica Typhimurium. EHEC colonizes the large intestine and produces a potent toxin, Shiga toxin (Stx), responsible for the hemorrhagic colitis and HUS, which can culminate in kidney failure and leads to the mortality associated with EHEC outbreaks (Kaper et al. 2004).

EHEC causes a histopathological lesion on intestinal epithelial cells called attaching and effacing (AE). The AE lesion is characterized by the destruction of the microvilli and the rearrangement of the cytoskeleton to form a unique pedestal structure that cups the bacterium individually (Fig. 12.1a).

Chemical signaling through the AI-3/Epinephrine/Norepineprhine signals activates expression of virulence genes in EHEC. Most of these virulence genes are involved in the formation of the AE lesion and are contained within a pathogenicity island named the locus of enterocyte effacement (LEE) (McDaniel et al. 1995) (Fig. 12.2).

The EHEC LEE region contains 41 genes, most of which are organized into five major operons: LEE1, LEE2, LEE3, LEE5, and LEE4 (Elliott et al. 1998; Elliott et al. 1999; Mellies et al. 1999). The LEE encodes a type III secretion system

Molecular Microbiology Department, University of Texas Southwestern Medical Center, 6000 Harry Hines Bvld, Dallas 75390, TX, USA e-mail: [email protected]

M. Lyte and P.P.E. Freestone (eds.), Microbial Endocrinology, Interkingdom Signaling in Infectious Disease and Health,

DOI 10.1007/978-1-4419-5576-0_12, © Springer Science+Business Media, LLC 2010

orf1 espG ler escRSTU ^ grlRA cesD escCJ sepZ escVN sepO ^ map tir cesT eae^ escD sepL espAD^F

Ler (Transcription activator) TTSS (Type 3 secretion system)

Tir (Translocated intimin receptor) Esps (Secreted proteins)

Fig. 12.1 (a) The locus of enterocyte effacement (LEE) pathogenicity Island found in EHEC, which encodes factors responsible for type III secretion and pedestal formation. LEE1 encodes for ler, the LEE-encoded regulator. LEE1, LEE2, and LEE3 encode for factors involved in type III secretion. LEE4 encodes for EspA, EspB, and EspD. The LEE5/tir operon encodes for intimin and Tir (McDaniel et al. 1995; Kenny et al. 1997a; Mellies et al. 1999)

Ehec Phage Lee

Fig. 12.2 General model for EHEC pathogenesis. LEE region of EHEC encodes most of T3SS effectors, which are essential for EHEC pathogenesis, as well as flagella motility. Ler is master regulator of LEE, intimin is an outer membrane protein, through T3SS EHEC translocates Tir, Intimin-Tir binding culminates in histopathologic lesion called attaching and effacing lesion (or lesion AE). Shiga toxin in EHEC plays late role during infection that can cause hemolictic uremic sindrome (Kaper et al. 2004)

Fig. 12.2 General model for EHEC pathogenesis. LEE region of EHEC encodes most of T3SS effectors, which are essential for EHEC pathogenesis, as well as flagella motility. Ler is master regulator of LEE, intimin is an outer membrane protein, through T3SS EHEC translocates Tir, Intimin-Tir binding culminates in histopathologic lesion called attaching and effacing lesion (or lesion AE). Shiga toxin in EHEC plays late role during infection that can cause hemolictic uremic sindrome (Kaper et al. 2004)

(TTSS) (Jarvis et al. 1995), an adhesin (intimin) (Jerse et al. 1990), and this adhesin's receptor, the translocated intimin receptor (Tir) (Kenny et al. 1997b), which is translocated into the epithelial cell through the bacterial TTSS (Elliott et al. 1998; Elliott et al. 1999; Mellies et al. 1999) (Fig. 12.1a).

The TTSS is an apparatus that spans the inner and outer bacterial membranes forming a microscopic "needle." Several proteins, including EscD, EscR, EscU, EscV, EscS, and EscT span the inner membrane and associate with a cytoplasmic ATPase, EscN, which is required for secretion of proteins (Roe et al. 2003). EscC is predicted to form the main protein ring in the outer membrane to which the EscF "needle" is connected (Wilson et al. 2001). EscF comprises the syringe connected to the filament of the translocon. The translocon consists of EspA, which creates a sheath around the EscF needle. EspB and EspD are located at the distal end of the TTSS and form 3-5 nm pores in the host cell membrane (Ide et al. 2001) through which translocated proteins are secreted.

The eae gene (E. coli attaching and effacing) encodes for intimin, an outer membrane protein that acts as an intestinal adherence factor (Jerse et al. 1990). Mutants of the eae gene are defective in intimate adherence to intestinal epithelial cells, which prevents the concentration of polymerized actin necessary for the development of AE lesions. The translocated intimin receptor (Tir), which is also encoded in the LEE, is translocated from the bacterium through the TTSS into the host cell to serve as a receptor for intimin (DeVinney et al. 1999; Kenny and Finlay 1995; Rosenshine et al. 1996). In the host cell membrane, Tir adopts a hairpin loop conformation and serves as a receptor for the bacterial surface adhesin, intimin (Deibel et al. 1998). Binding of intimin to Tir promotes the clustering of N- and C-terminal cytoplasmic regions and leads to the initiation of localized actin assembly beneath the plasma membrane (Campellone et al. 2004). The EHEC Tir recruits the host protein N-WASP (Goosney et al. 2001) through an interaction with EspFu, another bacterial protein encoded within a prophage, which is also translocated through the TTSS into the host cell (Campellone et al. 2004).

The TTSS encoded by the LEE translocates LEE-encoded and non-LEE encoded effectors. The mitochondrial associated protein, map, affects the integrity of the host mitochondrial membrane (Kenny and Jepson 2000) and is encoded directly upstream of tir. Another effector, EspF, is responsible for the disruption of the intestinal barrier function and induces cell death by an unknown mechanism (McNamara and Donnenberg 1998; McNamara et al. 2001). EspG is responsible for the disruption of microtubule formation and plays a role in virulence in the rabbit enteropathogenic E. coli (REPEC) model (Tomson et al. 2005), while EspH, which is encoded in LEE3, is responsible for the modulation of the host cell cytoskeleton through the inhibition of cell cycle signals (Tu et al. 2003). Although encoded outside the LEE pathogenicity island, several effector proteins have been recently shown to be secreted through the EHEC TTSS (Tobe et al. 2006). These include Cif, which induces host cell cycle arrest and reorganization of host actin cytoskeleton (Charpentier and Oswald 2004), and NleA, which has been shown to localize to the Golgi and play a key role in virulence in an animal model (Gruenheid et al. 2004).

EHEC also produces a powerful Shiga toxin (Stx) that is responsible for the major symptoms of hemorrhagic colitis and HUS. The Stx family contains two subgroups, Stx1 and Stx2. Stx1 shows little sequence variation between strains (Zhang et al. 2002), whereas antigenic divergence has been observed among the Stx2s, including Stx2, Stx2c, Stx2d, and Stx2e (Perera et al. 1988; Schmitt et al. 1991;

Zhang et al. 2002). Stx2 has been more associated epidemiological^ with severe human disease than Stx1 (Boerlin et al. 1999), with Stx2 and Stx2c being most frequently found in patients with HUS (Ritter et al. 1997).

The genes encoding Stx1 and Stx2 are located within the late genes of a l-like bacteriophage and are transcribed when the phage enters its lytic cycle (Neely and Friedman 1998). Once the phage replicates, Shiga toxin is produced, and the phage lyse the bacteria, thereby releasing the toxin into the host. The bacteriophage enters its lytic cycle during an SOS response triggered by disturbances in the bacterial membrane, DNA replication, or protein synthesis (Kimmitt et al. 1999; Kimmitt et al. 2000). These triggers are all common targets of conventional antibiotics and may contribute to the controversy surrounding the use of antibiotics to treat EHEC-mediated disease. Shiga toxins consist of a 1A:5B noncovalently associated subunit structure (Donohue-Rolfe et al. 1984). The B subunit of Stx is known to form a pentamer that binds to the eukaryotic glycolipid receptor, globotriaosylceramide (Jacewicz et al. 1986; Lindberg et al. 1987; Waddell et al. 1988). The A subunit is then cleaved by trypsin and reduced, resulting in a polypeptide that causes depuri-nation of a residue in the 28S rRNA of 60S ribosomes (Endo et al. 1988). This leads to the inhibition of protein synthesis, injury of renal glomerular endothelial cells, and the initiation of a pathophysiological cascade that leads to HUS (Fig. 12.2).

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