**4.6 Bundle-forming pilus (BFP)**

Typical EPEC strains produce a type IV pilus, the bundle-forming pili (BFP), which interconnects bacteria within microcolonies, promoting their stabilization and producing the LA phenotype [38]. The BFP is encoded by an operon of 14 genes contained on the EAF plasmid, with *bfpA* encoding the major structural subunit (bundlin) [54]. These 14 *bfp* genes are highly conserved among EPEC1 and EPEC2 strains. Some O128:H2 and O119:H2 EPEC strains that contain part of the *bfpA* gene have the rest of the *bfp* gene cluster deleted and replaced with an IS*66* element [55, 56].

### **4.7 The locus of enterocyte effacement (LEE) and the type III secretion system (TTSS)**

The locus of enterocyte effacement (LEE) is a 35.6-kb pathogenicity island of EPEC containing genes necessary for the formation of the A/E lesion [57]. The EPEC LEE contains at least 41 open reading frames that are organized into five operons

(*LEE1* to *LEE5*) [58–60]. *LEE1*, *LEE2*, and *LEE3* encode a type III protein secretion system (T3SS) and Ler (LEE-encoded regulator) regulators, such as GrlA (global regulator of LEE activator, formerly called Orf11) and GrlR (Grl repressor, formerly called Orf10) [61]. *LEE4* encodes the EPEC-secreted proteins EspA, EspB, and EspD via the type III system. LEE5 encodes intimin and its translocated receptor, Tir [62]. Besides Tir, the EPEC genome contains other six LEE-encoded effector proteins translocated into the cell (Map, EspF, EspG, EspZ, EspH, and EspB), which interfere with different aspects of the cell physiology ([58, 59]; reviewed in [44]) [63].

In addition to the LEE effectors, various non-LEE (Nle)-encoded effector genes (*cif*, *espI/nleA*, *nleB*, *nleC*, *nleD*, *nleE*, and *nleH*) [59, 63] were described, which are located outside the LEE region of EPEC, in at least six chromosomal PAIs, or in prophage elements (reviewed in [64] and [65]). Although they are not required for AE lesion formation, it is understood that they contribute to increased bacterial virulence [66].

The LEE region of some atypical EPEC strains display a genetic organization similar to that found in the typical EPEC prototype E2348/69 strain [66]. Although the T3SS-encoding genes are considerably conserved [66, 67], the effector proteinencoding genes display important differences, and remarkable differences can be detected at the 5′ and 3′ flanking regions of atypical EPEC, suggesting the occurrence of different evolution events [68].

The expression of LEE genes is controlled by Per, which is encoded on the EAF plasmid present in typical EPEC strains. Per activates Ler, which in turn activates the *LEE2*, *LEE3*, *LEE4*, and *LEE5* operons, and the genes *espF*, *espG*, and *map* [58, 59]. The Ler protein is a histone-like nucleoid-structuring protein (H-NS) that responds to an environmental stimulus (temperature). Ler also controls genes located outside the LEE, such as *espC* and *nleA* [60]. Additional regulatory system has been shown to control expression of the LEE [69]. The AI-2 (autoinducer-2) quorum-sensing system regulates LEE1 operon, which increases expression of the *LEE3* and *LEE4* operons via the *ler* gene product. Two novel LEE-encoded regulators that have roles in *ler* expression were reported, GrlA (global regulator of LEE activator) and GrlR (Grl repressor) [61]. GrlR and GrlA are positive and negative regulators, respectively, required for the expression of several LEE-encoded genes [61]. Other LEE regulators include the integration host factor (IHF); Bip, a tyrosine-phosphorylated GTPase; Fis (factor for inversion stimulation); and GadX, which is a member of the AraC transcription factor family [58].

#### **4.8 Intimin and Tir**

Intimin is a 94-KDa outer membrane adhesin encoded by the *eae* gene and required for intimate adherence of EPEC to epithelial cells at the sites of A/E lesions [24]. N-Terminal portions are highly conserved, whereas C-terminus portions are highly variable [70]. C-Terminal intimin differences have been used as a basis for classification into several distinct subtypes (represented by the Greek letters to α (alpha) through ζ (zeta) [71, 72]); human EPEC1 strains express subtype α, while EPEC2 strains express subtype β. The N-terminus portion binds intimin in the bacterial outer membrane, whereas the C-terminus portion binds intimin to Tir. The binding of intimin to Tir leads to intimate adherence of the bacterium to the epithelium and pedestal formation beneath adherent bacteria. In addition, Tir inhibits NF-қB activity by targeting tumor necrosis factor alpha (TNF-α) receptor-associated factors [73].

#### **4.9 Other potential adhesins**

In addition to BFP, two other EPEC surface structures, rodlike fimbriae and fibrillae, have been characterized and have been suggested to be involved in the

**57**

*Enteropathogenic Escherichia coli*

interactions [82].

**4.10 Flagella**

**5. EspC**

EPEC1 strains [24].

**6. Other toxins**

pathogenesis remains to be elucidated.

**7. Model of EPEC pathogenesis**

*DOI: http://dx.doi.org/10.5772/intechopen.82861*

interaction of EPEC with host cells [74, 75]. Additionally, EPEC strains encode a large surface protein, lymphocyte inhibitory factor (LifA), that contributes to epithelial cell adherence in vitro [76, 77] and is required for intestinal colonization of mice by the related A/E pathogen *C. rodentium* [78]. The *lifA* gene is more commonly found among typical rather than atypical EPEC strains [79]; however, atypical EPEC strains harboring *lifA* have a significant association with diarrhea in children under 5 years of age [80]. A novel gene cluster, designated the locus for diffuse adherence (*lda*), was found in an atypical EPEC O26 strain that is responsible for mediating DA adherence; its expression is induced by bile salts [81]. The *E. coli* common pilus (ECP) has also been shown to act as an accessory adherence factor in EPEC, playing a role during cell adherence and/or in bacterium-bacterium

Flagella has been suggested to be involved in EPEC adherence to epithelial cells [83]. EPEC mutants with transposon insertion in the flagellar gene *fliC* were deficient in localized adherence, and anti-flagellum antibodies were effective in blocking the adherence of several EPEC serotypes [83]. However, a subsequent study has

EspC is a high-molecular-weight secreted protein of EPEC that induces cytopathic effects on epithelial cells, including cytoskeletal damage [24, 85]. EspC is a member of the serine protease autotransporters of the *Enterobacteriaceae* (SPATE) family of autotransporter proteins that encodes its own transport mechanism. Moreover, espC has been shown to interact with and degrade hemoglobin [86] and to hydrolyze other proteins such as pepsin, factor V, and spectrin [87]. In addition, EspC confers enhanced lysozyme resistance to EPEC [87] and serves as a substratum for adherence and biofilm formation as well as to protect bacteria from antimicrobial compounds [88]. EspC is encoded in a 15-kb chromosomal island specific to

Scott and Kaper [89] reported a cytolethal distending toxin (CDT) in an EPEC

A three-stage model of EPEC pathogenesis was first described in the early 1990s [94], Clarke et al. [95], including localized adherence to the host cell, signal trans-

duction, and intimate attachment with pedestal formation (**Figure 2**).

strain that induces chromatin disruption, which leads to G2/M-phase growth arrest of the target cell and ultimately cell death [90]. A study has suggested that most EPEC strains from diarrhea harbor the CDT gene [91]. Another toxin is the enteroaggregative *E. coli* heat-stable enterotoxin 1 (EAST1) also present in EPEC strains [92]. The use of an EAST1 DNA probe suggests that this toxin is expressed by a number of clinical EPEC isolates [18, 93]. The role of CDT and EAST1 in EPEC

not confirmed a role of flagella in EPEC adherence [84].

*Enteropathogenic Escherichia coli DOI: http://dx.doi.org/10.5772/intechopen.82861*

interaction of EPEC with host cells [74, 75]. Additionally, EPEC strains encode a large surface protein, lymphocyte inhibitory factor (LifA), that contributes to epithelial cell adherence in vitro [76, 77] and is required for intestinal colonization of mice by the related A/E pathogen *C. rodentium* [78]. The *lifA* gene is more commonly found among typical rather than atypical EPEC strains [79]; however, atypical EPEC strains harboring *lifA* have a significant association with diarrhea in children under 5 years of age [80]. A novel gene cluster, designated the locus for diffuse adherence (*lda*), was found in an atypical EPEC O26 strain that is responsible for mediating DA adherence; its expression is induced by bile salts [81]. The *E. coli* common pilus (ECP) has also been shown to act as an accessory adherence factor in EPEC, playing a role during cell adherence and/or in bacterium-bacterium interactions [82].

#### **4.10 Flagella**

*The Universe of Escherichia coli*

rence of different evolution events [68].

AraC transcription factor family [58].

**4.8 Intimin and Tir**

**4.9 Other potential adhesins**

(*LEE1* to *LEE5*) [58–60]. *LEE1*, *LEE2*, and *LEE3* encode a type III protein secretion system (T3SS) and Ler (LEE-encoded regulator) regulators, such as GrlA (global regulator of LEE activator, formerly called Orf11) and GrlR (Grl repressor, formerly called Orf10) [61]. *LEE4* encodes the EPEC-secreted proteins EspA, EspB, and EspD via the type III system. LEE5 encodes intimin and its translocated receptor, Tir [62]. Besides Tir, the EPEC genome contains other six LEE-encoded effector proteins translocated into the cell (Map, EspF, EspG, EspZ, EspH, and EspB), which interfere

with different aspects of the cell physiology ([58, 59]; reviewed in [44]) [63].

In addition to the LEE effectors, various non-LEE (Nle)-encoded effector genes (*cif*, *espI/nleA*, *nleB*, *nleC*, *nleD*, *nleE*, and *nleH*) [59, 63] were described, which are located outside the LEE region of EPEC, in at least six chromosomal PAIs, or in prophage elements (reviewed in [64] and [65]). Although they are not required for AE lesion formation, it is understood that they contribute to increased bacterial virulence [66]. The LEE region of some atypical EPEC strains display a genetic organization similar to that found in the typical EPEC prototype E2348/69 strain [66]. Although the T3SS-encoding genes are considerably conserved [66, 67], the effector proteinencoding genes display important differences, and remarkable differences can be detected at the 5′ and 3′ flanking regions of atypical EPEC, suggesting the occur-

The expression of LEE genes is controlled by Per, which is encoded on the EAF plasmid present in typical EPEC strains. Per activates Ler, which in turn activates the *LEE2*, *LEE3*, *LEE4*, and *LEE5* operons, and the genes *espF*, *espG*, and *map* [58, 59]. The Ler protein is a histone-like nucleoid-structuring protein (H-NS) that responds to an environmental stimulus (temperature). Ler also controls genes located outside the LEE, such as *espC* and *nleA* [60]. Additional regulatory system has been shown to control expression of the LEE [69]. The AI-2 (autoinducer-2) quorum-sensing system regulates LEE1 operon, which increases expression of the *LEE3* and *LEE4* operons via the *ler* gene product. Two novel LEE-encoded regulators that have roles in *ler* expression were reported, GrlA (global regulator of LEE activator) and GrlR (Grl repressor) [61]. GrlR and GrlA are positive and negative regulators, respectively, required for the expression of several LEE-encoded genes [61]. Other LEE regulators include the integration host factor (IHF); Bip, a tyrosine-phosphorylated GTPase; Fis (factor for inversion stimulation); and GadX, which is a member of the

Intimin is a 94-KDa outer membrane adhesin encoded by the *eae* gene and required for intimate adherence of EPEC to epithelial cells at the sites of A/E lesions [24]. N-Terminal portions are highly conserved, whereas C-terminus portions are highly variable [70]. C-Terminal intimin differences have been used as a basis for classification into several distinct subtypes (represented by the Greek letters to α (alpha) through ζ (zeta) [71, 72]); human EPEC1 strains express subtype α, while EPEC2 strains express subtype β. The N-terminus portion binds intimin in the bacterial outer membrane, whereas the C-terminus portion binds intimin to Tir. The binding of intimin to Tir leads to intimate adherence of the bacterium to the epithelium and pedestal formation beneath adherent bacteria. In addition, Tir inhibits NF-қB activity by targeting tumor necrosis factor alpha (TNF-α) receptor-associated factors [73].

In addition to BFP, two other EPEC surface structures, rodlike fimbriae and fibrillae, have been characterized and have been suggested to be involved in the

**56**

Flagella has been suggested to be involved in EPEC adherence to epithelial cells [83]. EPEC mutants with transposon insertion in the flagellar gene *fliC* were deficient in localized adherence, and anti-flagellum antibodies were effective in blocking the adherence of several EPEC serotypes [83]. However, a subsequent study has not confirmed a role of flagella in EPEC adherence [84].

#### **5. EspC**

EspC is a high-molecular-weight secreted protein of EPEC that induces cytopathic effects on epithelial cells, including cytoskeletal damage [24, 85]. EspC is a member of the serine protease autotransporters of the *Enterobacteriaceae* (SPATE) family of autotransporter proteins that encodes its own transport mechanism. Moreover, espC has been shown to interact with and degrade hemoglobin [86] and to hydrolyze other proteins such as pepsin, factor V, and spectrin [87]. In addition, EspC confers enhanced lysozyme resistance to EPEC [87] and serves as a substratum for adherence and biofilm formation as well as to protect bacteria from antimicrobial compounds [88]. EspC is encoded in a 15-kb chromosomal island specific to EPEC1 strains [24].

#### **6. Other toxins**

Scott and Kaper [89] reported a cytolethal distending toxin (CDT) in an EPEC strain that induces chromatin disruption, which leads to G2/M-phase growth arrest of the target cell and ultimately cell death [90]. A study has suggested that most EPEC strains from diarrhea harbor the CDT gene [91]. Another toxin is the enteroaggregative *E. coli* heat-stable enterotoxin 1 (EAST1) also present in EPEC strains [92]. The use of an EAST1 DNA probe suggests that this toxin is expressed by a number of clinical EPEC isolates [18, 93]. The role of CDT and EAST1 in EPEC pathogenesis remains to be elucidated.

#### **7. Model of EPEC pathogenesis**

A three-stage model of EPEC pathogenesis was first described in the early 1990s [94], Clarke et al. [95], including localized adherence to the host cell, signal transduction, and intimate attachment with pedestal formation (**Figure 2**).

**Figure 2.** *Four-stage model of EPEC pathogenesis. Reprinted from Clarke et al. [95].*

In the first stage, an attachment of typical EPEC to the surface of the host intestinal epithelium is mediated by the bundle-forming pili (BFP). The filament EspA also promotes attachment, albeit in a less efficient manner, and could mediate adherence of atypical EPEC strains. In the second stage, Tir and effector proteins (EspB, EspD, EspF, EspG, and Map), translocated into the host cells via type III system apparatus, activate cell-signaling pathways, causing alterations in the host cell cytoskeleton and resulting in actin accumulation and loss of microvilli [58]. In the third stage, bacteria intimately adhered to host cell by intimin-Tir interactions amplifies the accumulation of filaments of actin and other cytoskeletal proteins that result in pedestal-like structures [62, 96, 97]. Finally, the translocated effectors disrupt host cell processes, causing loss of tight-junction integrity and mitochondrial function and leading to both electrolyte loss and eventual cell death.

## **8. Diagnosis**

Traditionally, the identification of EPEC was based on the O:H serotyping, but serotype designation is no longer precise. The identification of EPEC was based on the characteristic of EPEC's attachment to epithelial cells and may include phenotypic or genotypic tests. The HeLa adherence assay distinguishes EPEC from other *E. coli* by their ability to adhere in a localized pattern (LA) on the surface of cells [8]. The fluorescent actin-staining (FAS) assay, originally described by Knutton et al. [98], leads to the identification of the A/E lesion, by detecting actin condensation under EPEC adhesion pedestals. DNA probes and PCR targeting genes responsible for these characteristics were developed. A 1-kb EAF fragment probe was initially developed as a diagnostic DNA probe (the EAF probe) and subsequently refined as an oligonucleotide probe as well as PCR primers [10, 11, 99]. The identification of *bfpA*, the structural gene encoding BFP, led to the development of more specific and sensitive probe or PCR tests to detect typical EPEC strains [74, 75, 100]. However, some PCR primers may fail to identify all typical EPEC strains since multiple alleles of *bfpA* have been identified [101]. The *eae* sequences by DNA probes and PCR primers have been used to detect the presence of LEE encoding A/E lesion [24].

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*Enteropathogenic Escherichia coli*

**9.1 Symptoms**

**9.2 Treatment**

**9.3 Vaccines**

**10. Conclusion**

*DOI: http://dx.doi.org/10.5772/intechopen.82861*

**9. Clinical features of EPEC infection**

risk of death among infants in Kenya [24, 31, 97].

prevention of EPEC diarrhea [106].

The most common symptoms reported in EPEC infection are watery diarrhea, dehydration, vomiting, food intolerance, and low-grade fever [24, 97]. In addition, EPEC infection may lead to severe malabsorption of nutrients resulting in nutritional aggravation and persistence of diarrhea [102]. Edema, neutrophil infiltrate, and reduced enzymatic activity in the intestinal mucosa have been also found in EPEC infection [103]. EPEC diarrhea often lasts 1–2 weeks but can become persistent, lasting more than 2 weeks, and may result in severe infection [24, 25, 32, 102]. In a recent case-control study, EPEC infection was associated with a 2.8-fold elevated

Treatment of EPEC diarrhea includes oral rehydration therapy to prevent dehydration by correcting fluid and electrolyte losses. Oral rehydration may be sufficient for cases of self-limited acute diarrhea, but persistent cases of diarrhea may include parenteral rehydration, and more severe cases may require total parental nutrition and use of antimicrobials [102]. Multidrug resistance has been reported in EPEC strains from diverse parts of the world [27–29, 44, 104, 105]. Alternative therapies, employing the use of bismuth subsalicylate, specific bovine anti-EPEC milk immunoglobulins, and also zinc, have been proven useful for treatment and

There are no currently available vaccines to prevent EPEC infection. However, a recent study has used bacterial ghosts devoid of cytoplasmic contents but expressing all EPEC surface components in vaccination challenge experiments with mice, and the results showed 84–90% protection in control mice [107]. Interestingly, protective effect of breast-feeding was shown to provide excellent protection against EPEC infection. Several investigators have shown that breast milk provides protection against EPEC O antigens and outer membrane proteins [108, 109]. Furthermore, IgA antibodies against BFP, intimin, EspA, and EspB proteins were

Much progress has been made in the last 20 years toward understanding the pathogenesis of EPEC. It has been shown that typical EPEC are still important pathogens associated with severe outbreaks of infant diarrhea, and atypical EPEC are emerging pathogens associated with sporadic outbreaks at all ages worldwide.

identified in maternal colostrum and serum samples [110–118].
