Systematic Deletion of Type III Secretion System Effectors in Enteropathogenic *E. coli* Unveils the Role of Non-LEE Effectors in A/E Lesion Formation

*Massiel Cepeda-Molero, Stephanie Schüller, Gad Frankel and Luis Ángel Fernández*

#### **Abstract**

Enteropathogenic *E. coli* (EPEC) is a diarrheagenic human pathogen. The hallmark of EPEC infection is the formation of the attaching and effacing (A/E) lesion in the intestinal epithelial cells, characterized by the effacement of brush border microvilli and the intimate bacterial attachment to the enterocyte in actinrich pedestal-like structures. The locus of enterocyte effacement (LEE) in the EPEC genome encodes a type III protein secretion system (T3SS) that translocates multiple effector proteins into the host cell to subvert cellular functions for the benefit of the pathogen. These effectors are encoded both within and outside the LEE. In vitro cell culture infections have shown that LEE effectors are required for intimate bacterial attachment to the epithelial cells, whereas non-LEE effectors mostly play a role in modulating inflammation and cell apoptosis in the gut epithelium. We constructed a set of EPEC mutant strains harboring deletions in the complete repertoire of genes encoding T3SS effectors. Infection of human intestinal in vitro organ cultures (IVOC) with these mutant strains surprisingly revealed that non-LEE effectors are also needed to induce efficient A/E lesion formation in the intestinal mucosal tissue.

**Keywords:** A/E lesion, EPEC, effectors, infection, IVOC, T3SS

#### **1. Introduction**

#### **1.1 Enteropathogenic** *E. coli*

Enteropathogenic *E. coli* (EPEC) was the first pathotype of *E. coli* to be associated with human disease and is a major cause of acute and chronic diarrhea in infants [1, 2]. The low microbial density of the small bowel caused by the forceful peristalsis in this part of the intestine is overcome by EPEC, which can successfully colonize the small intestine of humans [3, 4]. EPEC primarily affects children younger than 2 years old; however some outbreaks of EPEC infection in healthy

adults have been associated with large inoculum ingestion [5]. The mechanism of transmission of EPEC is the fecal-oral route. In the 1940s and 1950s, EPEC was an important cause of diarrhea in developed countries with a mortality of 50% during outbreaks, but nowadays the infection by EPEC in industrial countries has a limited importance. In contrast, in low-income countries, EPEC is still an major cause of infant diarrhea [5, 6].

#### **1.2 Hallmark of EPEC gastrointestinal infection**

The phenotype that defines EPEC infection is the attaching and effacing (A/E) lesion [2, 7]. By adhering to intestinal epithelial cells, EPEC subverts cytoskeletal processes of the host cell and produces the histopathological feature of the A/E lesion. This lesion, which was first described in 1980 [8], is characterized by the intimate attachment of the bacteria to the intestinal epithelial cells and elongation and effacement of the brush border microvilli. Later on it was shown that infection is also associated with cytoskeletal rearrangements, including the accumulation of polymerized F-actin in pedestal-like structures underneath the attached bacteria [9] (**Figure 1**). EPEC together with enterohemorrhagic *E. coli* (EHEC) and *Citrobacter rodentium* (CR) is a member of the A/E family of bacterial pathogens that colonize the gastrointestinal tract via the A/E lesion. EPEC and EHEC are important human pathogens, while CR is a mouserestricted pathogen [10–13].

#### **Figure 1.**

*Localized adherence (LA), intimate attachment, and EPEC A/E lesion formation in the intestinal epithelial surface. At an early stage, EPEC interacts in a non-intimate manner with the intestinal surface mainly through the BFP and EspA filament. After assembly of the translocation pore, EPEC injects translocated intimin receptor (Tir). Ser/Thr phosphorylation of Tir induces its anchoring in the enterocyte plasma membrane, leaving the TirM region exposed for the interaction with intimin. Subsequent Tir-intimin interaction triggers actin polymerization and pedestal formation underneath the attached bacterium. Tir phosphorylation of residue Y474 engages the host adaptor NcK, which later recruits N-WASP and WIP. N-WASP recruits the ARP2/3 complex, which induces actin nucleation and polymerization.*

**81**

**Figure 2.**

*indicated at the bottom. Figure from [32].*

*Systematic Deletion of Type III Secretion System Effectors in Enteropathogenic* E. coli *Unveils…*

The ability of EPEC to induce A/E lesions is related to a pathogenicity island (PAI) of 35 kb called the locus of enterocyte effacement (LEE) [14]. The LEE comprises 41 genes organized in 5 principal operons (LEE1-LEE5) and several smaller transcriptional units (**Figure 2**) [15, 16]. Orthologues of LEE are also found in other members of A/E pathogens [11]. The LEE encodes all the structural proteins necessary for the assembly of a filamentous type III secretion system (T3SS) injectisome on the bacterial cell envelope [17, 18]. The LEE also encodes transcriptional regulators (Ler, GrlR, and GrlA), translocator proteins (EspA, EspB, and EspD), six secreted effector proteins (including the translocated intimin receptor), the outer membrane protein intimin, molecular chaperones, and a lytic transglycosylase (EtgA) [19]. The mechanism of LEE regulation is complex and depends on environmental conditions, quorum sensing (QS), and several transcriptional regulators encoded within and outside the

*Effectors of EPEC E2348/69. (A) Representation of the LEE island and effector genes espG, espZ, espH, map, tir, and espF. (B) Non-LEE effectors located outside the LEE are localized in integrative elements (IEs) and prophages (PPs). Effector genes are labeled in red. Pseudogenes are specified with asterisk. Scale of 5 kb is* 

*DOI: http://dx.doi.org/10.5772/intechopen.91677*

**2.1 A pathogenicity island called LEE**

**2. EPEC virulence factors**

LEE [20, 21].

*Systematic Deletion of Type III Secretion System Effectors in Enteropathogenic* E. coli *Unveils… DOI: http://dx.doi.org/10.5772/intechopen.91677*

#### **2. EPEC virulence factors**

E. Coli *Infections - Importance of Early Diagnosis and Efficient Treatment*

**1.2 Hallmark of EPEC gastrointestinal infection**

infant diarrhea [5, 6].

restricted pathogen [10–13].

adults have been associated with large inoculum ingestion [5]. The mechanism of transmission of EPEC is the fecal-oral route. In the 1940s and 1950s, EPEC was an important cause of diarrhea in developed countries with a mortality of 50% during outbreaks, but nowadays the infection by EPEC in industrial countries has a limited importance. In contrast, in low-income countries, EPEC is still an major cause of

The phenotype that defines EPEC infection is the attaching and effacing (A/E) lesion [2, 7]. By adhering to intestinal epithelial cells, EPEC subverts cytoskeletal processes of the host cell and produces the histopathological feature of the A/E lesion. This lesion, which was first described in 1980 [8], is characterized by the intimate attachment of the bacteria to the intestinal epithelial cells and elongation and effacement of the brush border microvilli. Later on it was shown that infection is also associated with cytoskeletal rearrangements, including the accumulation of polymerized F-actin in pedestal-like structures underneath the attached bacteria [9] (**Figure 1**). EPEC together with enterohemorrhagic *E. coli* (EHEC) and *Citrobacter rodentium* (CR) is a member of the A/E family of bacterial pathogens that colonize the gastrointestinal tract via the A/E lesion. EPEC and EHEC are important human pathogens, while CR is a mouse-

*Localized adherence (LA), intimate attachment, and EPEC A/E lesion formation in the intestinal epithelial surface. At an early stage, EPEC interacts in a non-intimate manner with the intestinal surface mainly through the BFP and EspA filament. After assembly of the translocation pore, EPEC injects translocated intimin receptor (Tir). Ser/Thr phosphorylation of Tir induces its anchoring in the enterocyte plasma membrane, leaving the TirM region exposed for the interaction with intimin. Subsequent Tir-intimin interaction triggers actin polymerization and pedestal formation underneath the attached bacterium. Tir phosphorylation of residue Y474 engages the host adaptor NcK, which later recruits N-WASP and WIP. N-WASP recruits the* 

*ARP2/3 complex, which induces actin nucleation and polymerization.*

**80**

**Figure 1.**

#### **2.1 A pathogenicity island called LEE**

The ability of EPEC to induce A/E lesions is related to a pathogenicity island (PAI) of 35 kb called the locus of enterocyte effacement (LEE) [14]. The LEE comprises 41 genes organized in 5 principal operons (LEE1-LEE5) and several smaller transcriptional units (**Figure 2**) [15, 16]. Orthologues of LEE are also found in other members of A/E pathogens [11]. The LEE encodes all the structural proteins necessary for the assembly of a filamentous type III secretion system (T3SS) injectisome on the bacterial cell envelope [17, 18]. The LEE also encodes transcriptional regulators (Ler, GrlR, and GrlA), translocator proteins (EspA, EspB, and EspD), six secreted effector proteins (including the translocated intimin receptor), the outer membrane protein intimin, molecular chaperones, and a lytic transglycosylase (EtgA) [19]. The mechanism of LEE regulation is complex and depends on environmental conditions, quorum sensing (QS), and several transcriptional regulators encoded within and outside the LEE [20, 21].

#### **Figure 2.**

*Effectors of EPEC E2348/69. (A) Representation of the LEE island and effector genes espG, espZ, espH, map, tir, and espF. (B) Non-LEE effectors located outside the LEE are localized in integrative elements (IEs) and prophages (PPs). Effector genes are labeled in red. Pseudogenes are specified with asterisk. Scale of 5 kb is indicated at the bottom. Figure from [32].*

#### **2.2 The type III secretion system**

The type III secretion system is a macromolecular transport apparatus that is used by many gram-negative bacterial pathogens (e.g., *Shigella*, *Yersinia*, *Salmonella*) to translocate virulence proteins, called effectors, into the cytosol of infected cells, thereby subverting host cellular functions for the benefit of the pathogen [22]. Since pathogens use this transport apparatus to inject proteins into the host cells, this structure is also known as the injectisome. The EPEC T3SS mediates the translocation of multiple effector proteins during infection. Some of them are encoded in the LEE, whereas others are encoded outside of the LEE being generally referred to as non-LEE effectors (Nle) [23, 24]. EspA filaments link the tip of the injectisome in the bacterial cell wall to a 3–5 nm translocation pore, formed in the plasma membrane of infected cells by the translocator proteins EspB and EspD (**Figure 1**) [25, 26].

#### **2.3 Bundle-forming pilus (BFP)**

Typical EPEC is endowed with a plasmid called pMAR2 which contains a 14-gene operon encoding the type IV pilus BFP [27, 28]. The BFP is a rope-like bundle, which allows EPEC to form microcolonies in a pattern called localized adherence and also mediates the initial interaction of bacteria with host cell surfaces (**Figure 1**) [29–31].

#### **3. EPEC pathogenesis**

EPEC tightly regulates its virulence genes in response to environmental conditions such as temperature [16], the increase of the pH of the small intestine [33, 34], and some hormones which are released during stress conditions [20]. Upon EPEC interaction with enterocytes, EspB and EspD proteins are inserted into the host cell membrane and assemble to form a translocation pore [25, 26]. EPEC then injects its own receptor called Tir, which is integrated into the plasma membrane in a hairpin loop topology, with the loop facing the outside of the cell where it serves as a receptor for the bacterial adhesin intimin [35–37]. Tir-intimin interaction induces clustering and dimerization of Tir, and this activates a signal cascade that starts with the phosphorylation of serine/threonine residues and leads to actin polymerization and pedestal formation underneath the attached bacterium [10, 38]. The most critical event for actin polymerization is the phosphorylation of the cytoplasmic Tir residue Y474 [39]. This induces a signal cascade which recruits the host cell adaptor Nck and N-WASP required to engage and activate the actin-nucleating ARP2/3 complex, which produces the actin nucleation and polymerization. Actin polymerization drives membrane protrusion and pedestal formation [10, 40] (**Figure 1**). Through the T3SS injectisome, EPEC translocates LEE-encoded effector proteins and additional effectors localized in mobile genetic element outside the LEE (Nle).

#### **4. LEE effectors**

Six effector proteins (EspG, EspZ, EspH, Map, Tir, and EspF) are encoded in the LEE island (**Figure 2**). Most of these, except EspZ, have important functions destabilizing the physiology of the intestinal epithelium, triggering cytoskeleton reorganization, inducing cytotoxicity and electrolyte imbalance which lead to

**83**

[52] (**Figure 3**).

**Figure 3.**

**5. Non-LEE effectors**

*Systematic Deletion of Type III Secretion System Effectors in Enteropathogenic* E. coli *Unveils…*

diarrhea [11, 41]. The rapid onset of EPEC-induced diarrhea is likely induced by the cooperative action of Tir, Map, and EspF, which inhibits the sodium-D-glucose transporter (SGLT-1), the major water pump of the small intestine responsible for about 70% of the total fluid uptake [42]. In addition, Map and EspF reduce Na + absorption by the sodium-hydrogen exchanger (NHE3) [43], and EspG1/2 proteins alter the membrane targeting of the Cl-/OH-exchanger (DRA), resulting in reduced Cl-uptake. These processes result in the accumulation of salts in the gut

*EPEC effector proteins altering epithelial cell function and inducing water loss and diarrhea. Tir, map, and EspF inhibit the sodium-D-glucose transporter. EspF reduces expression of the sodium-hydrogen exchanger NHE3. EspG and EspF induce mislocalization of aquaporins (AqP). EspG1/EspG2 alters membrane targeting of the Cl-/OH-exchanger. EspF, map, NleA, EspG1, and EspG2 disrupt tight junction complexes (TJ). EspB,* 

Inhibition of endosomal trafficking by EspG1/2 reduces the level of cell surface receptors [45]. In addition, EspF and EspG induce mislocalization of aquaporins (AQP), thereby reducing epithelial water absorption [46]. Furthermore, EspB, Tir, EspF, and Map induce microvillus effacement, and this reduction of absorptive surface likely exacerbates EPEC diarrhea [47]. While EspF and Map synergistically disrupt TJs [48], EspG1/2 induces microtubule disruption contributing to TJ disruption [49]. The effector protein NleA also disrupts TJs by blocking the delivery of new TJ proteins [49–51]. The disruption of TJs increases intestinal permeability and thereby likely contributes to EPEC-induced diarrhea

In EPEC prototype strain E2348/69, 17 functional Nle effectors are encoded in different integrative elements and prophages, frequently associated in gene clusters, with some effectors having duplicated gene copies and/or paralogs in different clusters [53] (**Figure 2**). EPEC infection is characterized by a weak inflammatory response [54]. Previous studies have shown that most Nle effectors and some LEE effectors inhibit the host immune response, which favors bacterial survival (**Figure 4**). Although NleF and NleH2 activate the NF-κB inflammatory pathway during early infection (ref), EPEC translocates several effectors that dampen the proinflammatory pathways of the cell [11]. Thus, a large number of Nle effectors inhibit host inflammation by different mechanisms, such as inhibition of the NF-κB (NleB, C, E, and H) and MAPK proinflammatory pathways (NleC and D) [55–58], inhibition

lumen, which drives water loss from the mucosa [44].

*DOI: http://dx.doi.org/10.5772/intechopen.91677*

*Tir, EspF, and map induce microvilli effacement.*

*Systematic Deletion of Type III Secretion System Effectors in Enteropathogenic* E. coli *Unveils… DOI: http://dx.doi.org/10.5772/intechopen.91677*

**Figure 3.**

E. Coli *Infections - Importance of Early Diagnosis and Efficient Treatment*

The type III secretion system is a macromolecular transport apparatus that is used by many gram-negative bacterial pathogens (e.g., *Shigella*, *Yersinia*, *Salmonella*) to translocate virulence proteins, called effectors, into the cytosol of infected cells, thereby subverting host cellular functions for the benefit of the pathogen [22]. Since pathogens use this transport apparatus to inject proteins into the host cells, this structure is also known as the injectisome. The EPEC T3SS mediates the translocation of multiple effector proteins during infection. Some of them are encoded in the LEE, whereas others are encoded outside of the LEE being generally referred to as non-LEE effectors (Nle) [23, 24]. EspA filaments link the tip of the injectisome in the bacterial cell wall to a 3–5 nm translocation pore, formed in the plasma membrane of infected cells by the translocator proteins EspB and EspD

Typical EPEC is endowed with a plasmid called pMAR2 which contains a 14-gene operon encoding the type IV pilus BFP [27, 28]. The BFP is a rope-like bundle, which allows EPEC to form microcolonies in a pattern called localized adherence and also mediates the initial interaction of bacteria with host cell surfaces

EPEC tightly regulates its virulence genes in response to environmental conditions such as temperature [16], the increase of the pH of the small intestine [33, 34], and some hormones which are released during stress conditions [20]. Upon EPEC interaction with enterocytes, EspB and EspD proteins are inserted into the host cell membrane and assemble to form a translocation pore [25, 26]. EPEC then injects its own receptor called Tir, which is integrated into the plasma membrane in a hairpin loop topology, with the loop facing the outside of the cell where it serves as a receptor for the bacterial adhesin intimin [35–37]. Tir-intimin interaction induces clustering and dimerization of Tir, and this activates a signal cascade that starts with the phosphorylation of serine/threonine residues and leads to actin polymerization and pedestal formation underneath the attached bacterium [10, 38]. The most critical event for actin polymerization is the phosphorylation of the cytoplasmic Tir residue Y474 [39]. This induces a signal cascade which recruits the host cell adaptor Nck and N-WASP required to engage and activate the actin-nucleating ARP2/3 complex, which produces the actin nucleation and polymerization. Actin polymerization drives membrane protrusion and pedestal formation [10, 40] (**Figure 1**). Through the T3SS injectisome, EPEC translocates LEE-encoded effector proteins and additional effectors localized in mobile genetic

Six effector proteins (EspG, EspZ, EspH, Map, Tir, and EspF) are encoded in the LEE island (**Figure 2**). Most of these, except EspZ, have important functions destabilizing the physiology of the intestinal epithelium, triggering cytoskeleton reorganization, inducing cytotoxicity and electrolyte imbalance which lead to

**2.2 The type III secretion system**

(**Figure 1**) [25, 26].

(**Figure 1**) [29–31].

**3. EPEC pathogenesis**

element outside the LEE (Nle).

**4. LEE effectors**

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

**82**

*EPEC effector proteins altering epithelial cell function and inducing water loss and diarrhea. Tir, map, and EspF inhibit the sodium-D-glucose transporter. EspF reduces expression of the sodium-hydrogen exchanger NHE3. EspG and EspF induce mislocalization of aquaporins (AqP). EspG1/EspG2 alters membrane targeting of the Cl-/OH-exchanger. EspF, map, NleA, EspG1, and EspG2 disrupt tight junction complexes (TJ). EspB, Tir, EspF, and map induce microvilli effacement.*

diarrhea [11, 41]. The rapid onset of EPEC-induced diarrhea is likely induced by the cooperative action of Tir, Map, and EspF, which inhibits the sodium-D-glucose transporter (SGLT-1), the major water pump of the small intestine responsible for about 70% of the total fluid uptake [42]. In addition, Map and EspF reduce Na + absorption by the sodium-hydrogen exchanger (NHE3) [43], and EspG1/2 proteins alter the membrane targeting of the Cl-/OH-exchanger (DRA), resulting in reduced Cl-uptake. These processes result in the accumulation of salts in the gut lumen, which drives water loss from the mucosa [44].

Inhibition of endosomal trafficking by EspG1/2 reduces the level of cell surface receptors [45]. In addition, EspF and EspG induce mislocalization of aquaporins (AQP), thereby reducing epithelial water absorption [46]. Furthermore, EspB, Tir, EspF, and Map induce microvillus effacement, and this reduction of absorptive surface likely exacerbates EPEC diarrhea [47]. While EspF and Map synergistically disrupt TJs [48], EspG1/2 induces microtubule disruption contributing to TJ disruption [49]. The effector protein NleA also disrupts TJs by blocking the delivery of new TJ proteins [49–51]. The disruption of TJs increases intestinal permeability and thereby likely contributes to EPEC-induced diarrhea [52] (**Figure 3**).

#### **5. Non-LEE effectors**

In EPEC prototype strain E2348/69, 17 functional Nle effectors are encoded in different integrative elements and prophages, frequently associated in gene clusters, with some effectors having duplicated gene copies and/or paralogs in different clusters [53] (**Figure 2**). EPEC infection is characterized by a weak inflammatory response [54]. Previous studies have shown that most Nle effectors and some LEE effectors inhibit the host immune response, which favors bacterial survival (**Figure 4**). Although NleF and NleH2 activate the NF-κB inflammatory pathway during early infection (ref), EPEC translocates several effectors that dampen the proinflammatory pathways of the cell [11]. Thus, a large number of Nle effectors inhibit host inflammation by different mechanisms, such as inhibition of the NF-κB (NleB, C, E, and H) and MAPK proinflammatory pathways (NleC and D) [55–58], inhibition

#### E. Coli *Infections - Importance of Early Diagnosis and Efficient Treatment*

#### **Figure 4.**

*Schematic representation of multifunctional and overlapping effectors to control host immune response. The NF-κB proinflammatory pathway is activated by NleF and NleH2 and is inhibited by NleE, NleB, NleH1, Tir, and NleC. NleC and NleD inhibit the MAPK proinflammatory pathway. EspF, EspJ, EspH, and EspB prevent macrophage phagocytosis. NleA disrupts inflammasome activation, and LifA inhibits IL-2 and IL-4 production and lymphocyte proliferation. While EspF and map induce intrinsic apoptosis, EspZ counteracts these effects by stabilizing mitochondrial membrane potential. NleH1/NleH2 and NleF inhibit intrinsic apoptosis, and NleF, NleD, and NleB counteract extrinsic apoptosis.*

of the canonical (NleA) and noncanonical (NleF) inflammasomes [59], and inhibition of proliferation of lymphocytes and interleukin production (LifA) [60, 61].

The control of the epithelial cell death response to microbial infection is pivotal for pathogens and the host. Pathogens that are colonizing the epithelium need to prevent cell death to preserve their replicative foothold; by contrast, the host needs to eliminate infected cells in order to minimize tissue damage [62]. During infection of the intestinal epithelial cells, surface properties of EPEC are recognized by cell surface death receptors and induce extrinsic apoptotic pathways, while T3SS effectors (Map and EspF) trigger cytochrome c release, activation of caspases, and downstream intrinsic apoptotic pathways [11, 24]. Interestingly, early stages of apoptosis can be observed during EPEC infection, but late stages are not evident because EPEC translocates effector proteins that antagonize these pro-apoptotic effects. NleD and NleB interfere with the pro-apoptotic death receptor signaling and disrupt the downstream extrinsic apoptosis [63, 64]. NLeH1/2 and EspZ also inhibit intrinsic apoptosis and promote host cell survival [65–67] (**Figure 4**). NleF directly inhibits caspases involved in both intrinsic and extrinsic apoptosis pathways, including caspases 4, 8, and 9 [68]. In addition, EspZ localizes to the cytoplasmic side of the plasma membrane at the site of bacterial attachment and interacts with the translocator protein EspD. It has been proposed that EspZ indirectly prevents cell death by downregulating protein translocation and protecting cells from an overdose of effector proteins. Consistently, a Δ*espZ* mutant was found to be highly cytotoxic [69]. EPEC effectors are injected in a regulated manner to guarantee the success of infection. While the pro-survival effector EspZ is translocated at the early stages of infection, the pro-apoptotic effectors EspF and Map follow later [70].

#### **6. Classical methodologies to study effector functions**

Most research on EPEC effectors has been conducted by generating deletion mutants in a single or a few effector genes that are later complemented with

**85**

**Figure 5.**

*Systematic Deletion of Type III Secretion System Effectors in Enteropathogenic* E. coli *Unveils…*

multicopy plasmids overexpressing the effector(s). In addition, ectopic expression of individual effectors by plasmid transfection of the host cell has been applied. Both situations are prone to effector overexpression resulting in nonphysiological levels of effectors inside the host cell, which could alter effector activities. In addition, effectors often have synergistic and overlapping functions that cannot be fully appreciated by single mutations and individual transfection experiments [11, 54]. In order to overcome these limitations, we employed a marker-less gene deletion strategy to delete the whole repertoire of known effector genes found in the genome of the prototypical EPEC strain E2348/69 [32]. The genome engineering method for sequential deletion of EPEC effectors was based on the marker-less gene deletion

Using this strategy, a set of EPEC mutants with sequential deletions of effectors was generated (**Table 1**), ultimately resulting in strains expressing only Tir and EspZ (EPEC2), Tir (EPEC1), and the effector-less strain EPEC0 (**Table 1**). This approach proved to be effective to specifically modify the genome of EPEC E2348/69, avoiding the introduction of unintended alterations in the genome and leaving no sequence "scars" or antibiotic resistance genes in the chromosome as demonstrated by whole-genome sequencing [32]. Besides, the deletion mutant strains showed normal growth and maintained functional T3SS injectisomes. In addition, they allowed the translocation of individual effectors from single-copy chromosomal genes under endogenous regulation, showing the expected phenotypes without the background of the other effectors [32]. Hence these mutant

*Marker-less gene deletion strategy of EPEC effector genes. Deletions using pGE-suicide plasmids with I-SceI sites and mutant alleles assembled by fusing homology regions (HRs) flanking the targeted effector gene(s). Co-integrants are identified by the Kanamycin resistance phenotype. Expression of the I-SceI in vivo from helper plasmid induces double-strand brakes that are repaired by homologous recombination. Depending on the HRs involved in this second recombination, either the WT or the mutant allele can be obtained. Figure from [32].*

technique described by Posfai et al. [71] and is illustrated in **Figure 5**.

*DOI: http://dx.doi.org/10.5772/intechopen.91677*

*Systematic Deletion of Type III Secretion System Effectors in Enteropathogenic* E. coli *Unveils… DOI: http://dx.doi.org/10.5772/intechopen.91677*

multicopy plasmids overexpressing the effector(s). In addition, ectopic expression of individual effectors by plasmid transfection of the host cell has been applied. Both situations are prone to effector overexpression resulting in nonphysiological levels of effectors inside the host cell, which could alter effector activities. In addition, effectors often have synergistic and overlapping functions that cannot be fully appreciated by single mutations and individual transfection experiments [11, 54]. In order to overcome these limitations, we employed a marker-less gene deletion strategy to delete the whole repertoire of known effector genes found in the genome of the prototypical EPEC strain E2348/69 [32]. The genome engineering method for sequential deletion of EPEC effectors was based on the marker-less gene deletion technique described by Posfai et al. [71] and is illustrated in **Figure 5**.

Using this strategy, a set of EPEC mutants with sequential deletions of effectors was generated (**Table 1**), ultimately resulting in strains expressing only Tir and EspZ (EPEC2), Tir (EPEC1), and the effector-less strain EPEC0 (**Table 1**). This approach proved to be effective to specifically modify the genome of EPEC E2348/69, avoiding the introduction of unintended alterations in the genome and leaving no sequence "scars" or antibiotic resistance genes in the chromosome as demonstrated by whole-genome sequencing [32]. Besides, the deletion mutant strains showed normal growth and maintained functional T3SS injectisomes. In addition, they allowed the translocation of individual effectors from single-copy chromosomal genes under endogenous regulation, showing the expected phenotypes without the background of the other effectors [32]. Hence these mutant

#### **Figure 5.**

E. Coli *Infections - Importance of Early Diagnosis and Efficient Treatment*

of the canonical (NleA) and noncanonical (NleF) inflammasomes [59], and inhibition of proliferation of lymphocytes and interleukin production (LifA) [60, 61].

*apoptosis, and NleF, NleD, and NleB counteract extrinsic apoptosis.*

*Schematic representation of multifunctional and overlapping effectors to control host immune response. The NF-κB proinflammatory pathway is activated by NleF and NleH2 and is inhibited by NleE, NleB, NleH1, Tir, and NleC. NleC and NleD inhibit the MAPK proinflammatory pathway. EspF, EspJ, EspH, and EspB prevent macrophage phagocytosis. NleA disrupts inflammasome activation, and LifA inhibits IL-2 and IL-4 production and lymphocyte proliferation. While EspF and map induce intrinsic apoptosis, EspZ counteracts these effects by stabilizing mitochondrial membrane potential. NleH1/NleH2 and NleF inhibit intrinsic* 

**6. Classical methodologies to study effector functions**

Most research on EPEC effectors has been conducted by generating deletion mutants in a single or a few effector genes that are later complemented with

The control of the epithelial cell death response to microbial infection is pivotal for pathogens and the host. Pathogens that are colonizing the epithelium need to prevent cell death to preserve their replicative foothold; by contrast, the host needs to eliminate infected cells in order to minimize tissue damage [62]. During infection of the intestinal epithelial cells, surface properties of EPEC are recognized by cell surface death receptors and induce extrinsic apoptotic pathways, while T3SS effectors (Map and EspF) trigger cytochrome c release, activation of caspases, and downstream intrinsic apoptotic pathways [11, 24]. Interestingly, early stages of apoptosis can be observed during EPEC infection, but late stages are not evident because EPEC translocates effector proteins that antagonize these pro-apoptotic effects. NleD and NleB interfere with the pro-apoptotic death receptor signaling and disrupt the downstream extrinsic apoptosis [63, 64]. NLeH1/2 and EspZ also inhibit intrinsic apoptosis and promote host cell survival [65–67] (**Figure 4**). NleF directly inhibits caspases involved in both intrinsic and extrinsic apoptosis pathways, including caspases 4, 8, and 9 [68]. In addition, EspZ localizes to the cytoplasmic side of the plasma membrane at the site of bacterial attachment and interacts with the translocator protein EspD. It has been proposed that EspZ indirectly prevents cell death by downregulating protein translocation and protecting cells from an overdose of effector proteins. Consistently, a Δ*espZ* mutant was found to be highly cytotoxic [69]. EPEC effectors are injected in a regulated manner to guarantee the success of infection. While the pro-survival effector EspZ is translocated at the early stages of infection, the pro-apoptotic effectors EspF and Map follow later [70].

**84**

**Figure 4.**

*Marker-less gene deletion strategy of EPEC effector genes. Deletions using pGE-suicide plasmids with I-SceI sites and mutant alleles assembled by fusing homology regions (HRs) flanking the targeted effector gene(s). Co-integrants are identified by the Kanamycin resistance phenotype. Expression of the I-SceI in vivo from helper plasmid induces double-strand brakes that are repaired by homologous recombination. Depending on the HRs involved in this second recombination, either the WT or the mutant allele can be obtained. Figure from [32].*


#### **Table 1.**

*EPEC mutant strains generated with the marker-less deletion strategy.*

strains are an excellent tool to investigate the role of individual effectors and specific combinations maintaining physiological protein levels in the context of infection.

#### **7. LEE effectors are sufficient for intimate adhesion of EPEC to the epithelial cells in vitro**

When EPEC bacteria adhere in vitro to cultured cells, there is an accumulation of actin filaments in the cytoplasm beneath the adherent bacteria, due to a signal cascade triggered by intimin-Tir interaction [35, 38]. Using the effector deletion

#### **Figure 6.**

*Infection of HeLa cells with EPEC WT and effector mutant strains. Immunofluorescence confocal microscopy of HeLa cells infected with EPEC WT, EPEC2, EPEC1, and EPEC0 for 1.5 h using a MOI of 200. EPEC is labeled with anti-intimin-280 serum (green), actin is stained with TRITC phalloidin (red), and cell nuclei are labeled with DAPI (gray). Actin polymerization beneath adherent bacteria is observed in EPEC WT, EPEC2, and EPEC1. Scale bar 5 μm. Figure from [32].*

**87**

*\**

**Table 2.**

*Encoded effectors in the indicated IEs and PPs.*

*Human duodenal biopsies infected by EPEC WT and EPEC effector mutants.*

*Systematic Deletion of Type III Secretion System Effectors in Enteropathogenic* E. coli *Unveils…*

mutants of EPEC, we demonstrated that the LEE effector Tir along with intimin is necessary and sufficient to induce these cytoskeletal rearrangements during in vitro infection of HeLa cells. Strains EPEC2 (bearing EspZ and Tir) and EPEC1 (bearing only Tir) were able to induce actin-pedestal formation underneath attached bacteria similar to the EPEC wild type (WT) (**Figure 6**). As expected because of the essential role of Tir in this process, infection of HeLa cells with the effector-less mutant EPEC0 did not induce any actin-pedestal formation (**Figure 6**). These data demonstrate that the individual translocation of Tir by EPEC1 is sufficient to trigger actin pedestals in HeLa cells and that non-LEE effectors are dispensable for this

**8. Non-LEE effectors are required for efficient A/E lesion formation in** 

EPEC pathogenic mechanisms have been widely investigated by in vitro infection of cultured epithelial cell lines, albeit in most cases these cells are non-polarized and are not from intestinal origin. In addition, EPEC infection studies in vivo are hindered because EPEC is a human-restricted pathogen [72]. A surrogate model established to investigate A/E pathogenesis in vivo is the mouse pathogen *Citrobacter rodentium* (CR) [12, 13]. Although *Citrobacter* infection in vitro requires Tir phosphorylation for actin-pedestal formation in cell lines, Tir phosphorylation-deficient mutants still colonize the mouse gut and induce A/E lesion formation and crypt hyperplasia typical of CR infection [73]. This result highlights the necessity of a model for EPEC infection closer to the in vivo conditions in the human gut. A good established model to study EPEC-host interactions is the infection of in vitro cultured human intestinal biopsies, which allows the formation of A/E lesions undistinguishable from those observed in vivo in biopsies of patients with EPEC-induced diarrhea [4, 36, 74]. Similar to results obtained in CR-infected mice, Tir phosphorylation was not necessary for EPEC A/E lesion formation in human intestinal biopsies [75]. Surprisingly, when EPEC2 and EPEC1 deletion mutants were used to infect human duodenal biopsies, none of the infected biopsies showed A/E lesions (**Table 2** and **Figure 7**), which contrasts with the pedestal formation observed in HeLa cells. Thus, intimin and Tir are not sufficient to induce A/E lesions in the intestinal tissue, and the IVOC model was used to identify additional LEE or non-LEE effector(s) required for A/E lesion formation. For this purpose, two additional effector mutant strains were tested: EPEC2-LEE+ (carrying all LEE effectors) and EPEC9 (carrying EspZ, Tir, and

**Strain Effector genes remaining\* Biopsies with A/E lesions** 

WT All 13/17 (76) EPEC2 *espZ* and *tir* 0/6 (0) EPEC1 *tir* 0/6 (0) EPEC0 None 0/5 (0) EPEC9 *espZ + tir + IE2 + IE5 + IE6 + PP2 + PP3 + PP4 + PP6* 5/6 (83) EPEC2-LEE+ *espZ + tir + map + espH + espF + espG* 0/5 (0)

**positive/total (%)**

*DOI: http://dx.doi.org/10.5772/intechopen.91677*

phenotype during in vitro infection of cultured cells.

**intestinal tissue**

*Systematic Deletion of Type III Secretion System Effectors in Enteropathogenic* E. coli *Unveils… DOI: http://dx.doi.org/10.5772/intechopen.91677*

mutants of EPEC, we demonstrated that the LEE effector Tir along with intimin is necessary and sufficient to induce these cytoskeletal rearrangements during in vitro infection of HeLa cells. Strains EPEC2 (bearing EspZ and Tir) and EPEC1 (bearing only Tir) were able to induce actin-pedestal formation underneath attached bacteria similar to the EPEC wild type (WT) (**Figure 6**). As expected because of the essential role of Tir in this process, infection of HeLa cells with the effector-less mutant EPEC0 did not induce any actin-pedestal formation (**Figure 6**). These data demonstrate that the individual translocation of Tir by EPEC1 is sufficient to trigger actin pedestals in HeLa cells and that non-LEE effectors are dispensable for this phenotype during in vitro infection of cultured cells.

#### **8. Non-LEE effectors are required for efficient A/E lesion formation in intestinal tissue**

EPEC pathogenic mechanisms have been widely investigated by in vitro infection of cultured epithelial cell lines, albeit in most cases these cells are non-polarized and are not from intestinal origin. In addition, EPEC infection studies in vivo are hindered because EPEC is a human-restricted pathogen [72]. A surrogate model established to investigate A/E pathogenesis in vivo is the mouse pathogen *Citrobacter rodentium* (CR) [12, 13]. Although *Citrobacter* infection in vitro requires Tir phosphorylation for actin-pedestal formation in cell lines, Tir phosphorylation-deficient mutants still colonize the mouse gut and induce A/E lesion formation and crypt hyperplasia typical of CR infection [73]. This result highlights the necessity of a model for EPEC infection closer to the in vivo conditions in the human gut. A good established model to study EPEC-host interactions is the infection of in vitro cultured human intestinal biopsies, which allows the formation of A/E lesions undistinguishable from those observed in vivo in biopsies of patients with EPEC-induced diarrhea [4, 36, 74]. Similar to results obtained in CR-infected mice, Tir phosphorylation was not necessary for EPEC A/E lesion formation in human intestinal biopsies [75]. Surprisingly, when EPEC2 and EPEC1 deletion mutants were used to infect human duodenal biopsies, none of the infected biopsies showed A/E lesions (**Table 2** and **Figure 7**), which contrasts with the pedestal formation observed in HeLa cells. Thus, intimin and Tir are not sufficient to induce A/E lesions in the intestinal tissue, and the IVOC model was used to identify additional LEE or non-LEE effector(s) required for A/E lesion formation. For this purpose, two additional effector mutant strains were tested: EPEC2-LEE+ (carrying all LEE effectors) and EPEC9 (carrying EspZ, Tir, and


#### **Table 2.**

*Human duodenal biopsies infected by EPEC WT and EPEC effector mutants.*

E. Coli *Infections - Importance of Early Diagnosis and Efficient Treatment*

**Strain Effector genes remaining\***

WT All

EPEC1 *tir* EPEC0 None

*Encoded effectors in the indicated IEs and PPs.*

EPEC2 *espZ* and *tir*

strains are an excellent tool to investigate the role of individual effectors and specific combinations maintaining physiological protein levels in the context of infection.

When EPEC bacteria adhere in vitro to cultured cells, there is an accumulation of actin filaments in the cytoplasm beneath the adherent bacteria, due to a signal cascade triggered by intimin-Tir interaction [35, 38]. Using the effector deletion

**7. LEE effectors are sufficient for intimate adhesion of EPEC to the** 

EPEC9 *espZ + tir + IE2 + IE5 + IE6 + PP2 + PP3 + PP4 + PP6*

EPEC2-LEE+ *espZ + tir + map + espH + espF + espG*

*EPEC mutant strains generated with the marker-less deletion strategy.*

*Infection of HeLa cells with EPEC WT and effector mutant strains. Immunofluorescence confocal microscopy of HeLa cells infected with EPEC WT, EPEC2, EPEC1, and EPEC0 for 1.5 h using a MOI of 200. EPEC is labeled with anti-intimin-280 serum (green), actin is stained with TRITC phalloidin (red), and cell nuclei are labeled with DAPI (gray). Actin polymerization beneath adherent bacteria is observed in EPEC* 

*WT, EPEC2, and EPEC1. Scale bar 5 μm. Figure from [32].*

**epithelial cells in vitro**

*\**

**Table 1.**

**86**

**Figure 6.**

#### **Figure 7.**

*Scanning electron microscopy of human duodenal biopsies infected with EPEC WT and mutant strains EPEC2, EPEC1, EPEC0, EPEC9, and EPEC2-LEE+. EPEC WT and EPEC9 induce characteristic A/E lesions with bacterial microcolony formation (asterisk) and microvilli elongation around bacterial colonies (arrowheads). In contrast, biopsies infected with EPEC2, EPEC1, EPEC0, and EPEC2-LEE+ lack adherent bacteria and A/E lesions and show a normal microvillous brush border. Scale bar 2 μm. Figure from [32].*

all non-LEE effectors). Whereas infection with EPEC2-LEE+ did not reveal A/E lesions, infection with EPEC9 induced A/E lesions to a similar level as the wildtype strain (**Table 2** and **Figure 7**). It was previously reported that the LEE island is sufficient to confer the A/E phenotype to *E. coli* K-12 in the infection of colon carcinoma cell lines [76]. However, our results indicate that the LEE is not sufficient for A/E lesion formation in human mucosal tissue and that non-LEE effectors are required [32].

#### **9. Conclusions and future perspectives**

The marker-less gene deletion strategy enabled the generation of effector-less strains of EPEC O127:H6 using the prototypical strain E2348/69 [32]. Given the conservation of the recombination machinery among *E. coli* strains, it is likely that

**89**

tinal tract [81].

**Acknowledgements**

Research (URICI).

*Systematic Deletion of Type III Secretion System Effectors in Enteropathogenic* E. coli *Unveils…*

this strategy could be applied to other A/E pathogens, *E. coli* pathogens, and other bacteria. The effector mutant strains can be useful to study the role of individual effectors and of combinations of effectors in pathogenesis. An individual effector or a defined combination can be inserted in the effector-less strains in their endogenous genomic loci to obtain physiological expression levels and regulation. In cell culture infections, all EPEC effector mutant strains carrying intimin and Tir were able to trigger actin-rich pedestal-like structures underneath attached bacteria. On the other hand, when the infection was performed in human intestinal tissues, translocation of Tir alone was insufficient to induce A/E lesions. Furthermore, an EPEC deletion mutant maintaining all LEE effectors and devoid of all non-LEE effectors (EPEC2-LEE+) was still unable to induce A/E lesions in human intestinal biopsies. In contrast, an EPEC strain producing the complete repertoire of non-LEE effectors and devoid of LEE effectors, except Tir and EspZ, formed A/E lesions in intestinal tissue at wild-type levels [32]. Thus, these experiments revealed that non-LEE effectors are needed for A/E lesion formation in human intestinal tissue. In addition to their potential for basic studies, the EPEC effector mutant strains may have different applications. For instance, EPEC (and other pathogenic) strains lacking multiple effectors are likely to be strongly attenuated, but they maintain the external antigenicity of the wild-type strain. Thus, an EPEC mutant strain with a functional T3SS and the minimum set of effectors necessary to colonize the intestinal surface could be a good vaccine candidate. Further, EPEC mutant strains with the ability to attach to the human intestine could also be engineered to translocate heterologous protein antigens to generate protection against other enteric pathogens causing diarrhea, including EHEC strains [77–80]. Lastly, the EPEC effector mutant strains may also have the potential to deliver therapeutic proteins to the intestinal epithelium, for instance, to combat inflammation and autoimmune disorders in the gastrointes-

We acknowledge support for the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for

*DOI: http://dx.doi.org/10.5772/intechopen.91677*

#### *Systematic Deletion of Type III Secretion System Effectors in Enteropathogenic* E. coli *Unveils… DOI: http://dx.doi.org/10.5772/intechopen.91677*

this strategy could be applied to other A/E pathogens, *E. coli* pathogens, and other bacteria. The effector mutant strains can be useful to study the role of individual effectors and of combinations of effectors in pathogenesis. An individual effector or a defined combination can be inserted in the effector-less strains in their endogenous genomic loci to obtain physiological expression levels and regulation. In cell culture infections, all EPEC effector mutant strains carrying intimin and Tir were able to trigger actin-rich pedestal-like structures underneath attached bacteria. On the other hand, when the infection was performed in human intestinal tissues, translocation of Tir alone was insufficient to induce A/E lesions. Furthermore, an EPEC deletion mutant maintaining all LEE effectors and devoid of all non-LEE effectors (EPEC2-LEE+) was still unable to induce A/E lesions in human intestinal biopsies. In contrast, an EPEC strain producing the complete repertoire of non-LEE effectors and devoid of LEE effectors, except Tir and EspZ, formed A/E lesions in intestinal tissue at wild-type levels [32]. Thus, these experiments revealed that non-LEE effectors are needed for A/E lesion formation in human intestinal tissue.

In addition to their potential for basic studies, the EPEC effector mutant strains may have different applications. For instance, EPEC (and other pathogenic) strains lacking multiple effectors are likely to be strongly attenuated, but they maintain the external antigenicity of the wild-type strain. Thus, an EPEC mutant strain with a functional T3SS and the minimum set of effectors necessary to colonize the intestinal surface could be a good vaccine candidate. Further, EPEC mutant strains with the ability to attach to the human intestine could also be engineered to translocate heterologous protein antigens to generate protection against other enteric pathogens causing diarrhea, including EHEC strains [77–80]. Lastly, the EPEC effector mutant strains may also have the potential to deliver therapeutic proteins to the intestinal epithelium, for instance, to combat inflammation and autoimmune disorders in the gastrointestinal tract [81].

#### **Acknowledgements**

E. Coli *Infections - Importance of Early Diagnosis and Efficient Treatment*

all non-LEE effectors). Whereas infection with EPEC2-LEE+ did not reveal A/E lesions, infection with EPEC9 induced A/E lesions to a similar level as the wildtype strain (**Table 2** and **Figure 7**). It was previously reported that the LEE island is sufficient to confer the A/E phenotype to *E. coli* K-12 in the infection of colon carcinoma cell lines [76]. However, our results indicate that the LEE is not sufficient for A/E lesion formation in human mucosal tissue and that non-LEE effectors

*lesions and show a normal microvillous brush border. Scale bar 2 μm. Figure from [32].*

*Scanning electron microscopy of human duodenal biopsies infected with EPEC WT and mutant strains EPEC2, EPEC1, EPEC0, EPEC9, and EPEC2-LEE+. EPEC WT and EPEC9 induce characteristic A/E lesions with bacterial microcolony formation (asterisk) and microvilli elongation around bacterial colonies (arrowheads). In contrast, biopsies infected with EPEC2, EPEC1, EPEC0, and EPEC2-LEE+ lack adherent bacteria and A/E* 

The marker-less gene deletion strategy enabled the generation of effector-less strains of EPEC O127:H6 using the prototypical strain E2348/69 [32]. Given the conservation of the recombination machinery among *E. coli* strains, it is likely that

**88**

are required [32].

**Figure 7.**

**9. Conclusions and future perspectives**

We acknowledge support for the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI).

#### **Author details**

Massiel Cepeda-Molero1 , Stephanie Schüller2 , Gad Frankel3 and Luis Ángel Fernández1 \*

1 Department of Microbial Biotechnology, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas (CSIC), Madrid, Spain

2 Norwich Medical School, University of East Anglia, Norwich, United Kingdom

3 Department of Life Sciences, MRC Centre for Molecular Bacteriology and Infection, Imperial College, London, United Kingdom

\*Address all correspondence to: lafdez@cnb.csic.es

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**91**

*Systematic Deletion of Type III Secretion System Effectors in Enteropathogenic* E. coli *Unveils…*

*coli*. Infection and Immunity.

[10] Lai Y et al. Intimate host attachment: Enteropathogenic and enterohaemorrhagic *Escherichia coli*. Cellular Microbiology. 2013;**15**(11):1796-1808

*coli*: Even more subversive

2011;**80**(6):1420-1438

[11] Wong AR et al. Enteropathogenic and enterohaemorrhagic *Escherichia* 

elements. Molecular Microbiology.

[12] Collins JW et al. Citrobacter rodentium: Infection, inflammation and the microbiota. Nature Reviews. Microbiology. 2014;**12**(9):612-623

[13] Mundy R et al. Citrobacter rodentium of mice and man. Cellular Microbiology. 2005;**7**(12):1697-1706

[15] Elliott SJ et al. The locus of

[14] McDaniel TK et al. A genetic locus of enterocyte effacement conserved among diverse enterobacterial

pathogens. Proceedings of the National Academy of Sciences of the United States of America. 1995;**92**(5):1664-1668

enterocyte effacement (LEE)-encoded regulator controls expression of both LEE- and non-LEE-encoded virulence factors in enteropathogenic and enterohemorrhagic *Escherichia coli*. Infection and Immunity. 2000;**68**(11):6115-6126

[16] Yerushalmi G et al. Dynamics of expression and maturation of the type III secretion system of enteropathogenic *Escherichia coli*. Journal of Bacteriology.

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*DOI: http://dx.doi.org/10.5772/intechopen.91677*

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**Author details**

Massiel Cepeda-Molero1

and Luis Ángel Fernández1

, Stephanie Schüller2

1 Department of Microbial Biotechnology, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas (CSIC), Madrid, Spain

2 Norwich Medical School, University of East Anglia, Norwich, United Kingdom

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

3 Department of Life Sciences, MRC Centre for Molecular Bacteriology and

\*

Infection, Imperial College, London, United Kingdom

\*Address all correspondence to: lafdez@cnb.csic.es

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[53] Iguchi A et al. Complete genome sequence and comparative genome analysis of enteropathogenic *Escherichia coli* O127:H6 strain E2348/69. Journal of Bacteriology. 2009;**191**(1):347-354

[54] Dean P, Kenny B. The effector repertoire of enteropathogenic *E. coli*: Ganging up on the host cell. Current Opinion in Microbiology. 2009;**12**(1):101-109

[55] Baruch K et al. Metalloprotease type III effectors that specifically cleave JNK and NF-kappaB. The EMBO Journal. 2011;**30**(1):221-231

[56] Nadler C et al. The type III secretion effector NleE inhibits NF-kappaB activation. PLoS Pathogens. 2010;**6**(1):e1000743

[57] Sham HP et al. Attaching and effacing bacterial effector NleC suppresses epithelial inflammatory responses by inhibiting NF-{kappa} B and p38 mitogen-activated protein kinase activation. Infection and Immunity. 2011;**79**(9):3552-3562

[58] Gao X et al. NleB, a bacterial effector with glycosyltransferase activity, targets GADPH function to inhibit NF-κB activation. Cell Host & Microbe. 2013;**13**(1):87-99

[59] Yen H, Sugimoto N, Tobe T. Enteropathogenic *Escherichia coli* uses NleA to inhibit NLRP3 inflammasome activation. PLoS Pathogens. 2015;**11**(9):e1005121

[60] Abu-Median AB et al. Functional analysis of lymphostatin homologues in enterohaemorrhagic *Escherichia coli*. FEMS Microbiology Letters. 2006;**258**(1):43-49

[61] Klapproth JM et al. A large toxin from pathogenic *Escherichia coli* strains that inhibits lymphocyte activation. Infection and Immunity. 2000;**68**(4):2148-2155

[62] Kim M et al. Bacterial interactions with the host epithelium. Cell Host & Microbe. 2010;**8**(1):20-35

[63] Pearson JS et al. A type III effector antagonizes death receptor signalling during bacterial gut infection. Nature. 2013;**501**(7466):247-251

[64] Wong Fok Lung T et al. The cell death response to enteropathogenic *Escherichia coli* infection. Cellular Microbiology. 2014;**16**(12):1736-1745

[65] Roxas JL et al. The enteropathogenic *Escherichia coli*-secreted protein EspZ inhibits host cell apoptosis. Infection and Immunity. 2012;**80**(11):3850-3857

[66] Hemrajani C et al. NleH effectors interact with Bax inhibitor-1 to block apoptosis during enteropathogenic *Escherichia coli* infection. Proceedings of the National Academy of Sciences. 2010;**107**(7):3129-3134

[67] Royan SV et al. Enteropathogenic *E. coli* non-LEE encoded effectors NleH1 and NleH2 attenuate NF-kappaB activation. Molecular Microbiology. 2010;**78**(5):1232-1245

[68] Blasche S et al. The *E. coli* effector protein NleF is a Caspase inhibitor. PLoS One. 2013;**8**(3):e58937

[69] Berger CN et al. EspZ of enteropathogenic and enterohemorrhagic *Escherichia coli* regulates type III secretion system protein translocation. MBio. 2012;**3**(5):e00317-12

**95**

*Systematic Deletion of Type III Secretion System Effectors in Enteropathogenic* E. coli *Unveils…*

(efa-1′) confers protective immunity to mice infected with *E. coli* O157:H7. Frontiers in Cellular and Infection Microbiology. 2016;**5**:104-104

[78] Szu SC, Ahmed A. Clinical studies of *Escherichia coli* O157:H7 conjugate vaccines in adults and young children. Microbiology Spectrum. 2014;**2**(6):1-7

[79] Rabinovitz BC et al. The intranasal vaccination of pregnant dams with Intimin and EspB confers protection in neonatal mice from *Escherichia coli* (EHEC) O157:H7 infection. Vaccine.

[80] Marcato P et al. Recombinant Shiga toxin B-subunit-keyhole limpet hemocyanin conjugate vaccine protects mice from Shigatoxemia. Infection and Immunity. 2005;**73**(10):6523-6529

Biotechnology. 2015;**35**:94-102

[81] Piñero-Lambea C, Ruano-Gallego D, Fernández LÁ. Engineered bacteria as therapeutic agents. Current Opinion in

2016;**34**(25):2793-2797

*DOI: http://dx.doi.org/10.5772/intechopen.91677*

[70] Mills E et al. Real-time analysis of effector translocation by the type III secretion system of enteropathogenic *Escherichia coli*. Cell Host & Microbe.

[71] Posfai G et al. Markerless gene replacement in *Escherichia coli* stimulated by a double-strand break in the chromosome. Nucleic Acids Research. 1999;**27**(22):4409-4415

Walker-Smith JA. Enteropathogenic *Escherichia coli* and life threatening

[74] Knutton S, Lloyd DR, McNeish AS. Adhesion of enteropathogenic *Escherichia coli* to human intestinal enterocytes and cultured human intestinal mucosa. Infection and Immunity.

[75] Schüller S et al. Tir phosphorylation

and Nck/N-WASP recruitment by enteropathogenic and

[76] McDaniel TK, Kaper JB. A cloned pathogenicity island from enteropathogenic *Escherichia coli* confers the attaching and effacing phenotype on *E. coli* K-12. Molecular Microbiology.

with DNA encoding truncated enterohemorrhagic *Escherichia coli* (EHEC) factor for adherence-1 gene

[77] Riquelme-Neira R et al. Vaccination

enterohaemorrhagic *Escherichia coli* during ex vivo colonization of human intestinal mucosa is different to cell culture models. Cellular Microbiology.

2008;**3**(2):104-113

[72] Hill SM, Phillips AD,

chronic diarrhoea. Gut. 1991;**32**(2):154-158

1987;**55**(1):69-77

2007;**9**(5):1352-1364

1997;**23**(2):399-407

[73] Deng W et al. Citrobacter rodentium translocated intimin receptor (Tir) is an essential virulence factor needed for actin condensation, intestinal colonization and colonic hyperplasia in mice. Molecular Microbiology. 2003;**48**(1):95-115

*Systematic Deletion of Type III Secretion System Effectors in Enteropathogenic* E. coli *Unveils… DOI: http://dx.doi.org/10.5772/intechopen.91677*

[70] Mills E et al. Real-time analysis of effector translocation by the type III secretion system of enteropathogenic *Escherichia coli*. Cell Host & Microbe. 2008;**3**(2):104-113

E. Coli *Infections - Importance of Early Diagnosis and Efficient Treatment*

[60] Abu-Median AB et al. Functional analysis of lymphostatin homologues in enterohaemorrhagic *Escherichia coli*. FEMS Microbiology Letters.

[62] Kim M et al. Bacterial interactions with the host epithelium. Cell Host &

[63] Pearson JS et al. A type III effector antagonizes death receptor signalling during bacterial gut infection. Nature.

[64] Wong Fok Lung T et al. The cell death response to enteropathogenic *Escherichia coli* infection. Cellular Microbiology. 2014;**16**(12):1736-1745

[65] Roxas JL et al. The enteropathogenic *Escherichia coli*-secreted protein EspZ inhibits host cell apoptosis. Infection and Immunity. 2012;**80**(11):3850-3857

[66] Hemrajani C et al. NleH effectors interact with Bax inhibitor-1 to block apoptosis during enteropathogenic *Escherichia coli* infection. Proceedings of the National Academy of Sciences.

[67] Royan SV et al. Enteropathogenic *E. coli* non-LEE encoded effectors NleH1 and NleH2 attenuate NF-kappaB activation. Molecular Microbiology.

[68] Blasche S et al. The *E. coli* effector protein NleF is a Caspase inhibitor. PLoS One. 2013;**8**(3):e58937

[61] Klapproth JM et al. A large toxin from pathogenic *Escherichia coli* strains that inhibits lymphocyte activation. Infection and Immunity.

2006;**258**(1):43-49

2000;**68**(4):2148-2155

Microbe. 2010;**8**(1):20-35

2013;**501**(7466):247-251

2010;**107**(7):3129-3134

2010;**78**(5):1232-1245

[69] Berger CN et al. EspZ of enteropathogenic and enterohemorrhagic *Escherichia coli* regulates type III secretion system protein translocation. MBio.

2012;**3**(5):e00317-12

[51] Kim J et al. The bacterial virulence factor NleA inhibits cellular protein secretion by disrupting mammalian COPII function. Cell Host & Microbe.

[52] Croxen MA, Finlay BB. Molecular

[53] Iguchi A et al. Complete genome sequence and comparative genome analysis of enteropathogenic *Escherichia coli* O127:H6 strain E2348/69. Journal of Bacteriology. 2009;**191**(1):347-354

[54] Dean P, Kenny B. The effector repertoire of enteropathogenic *E. coli*: Ganging up on the host cell. Current Opinion in Microbiology.

[55] Baruch K et al. Metalloprotease type III effectors that specifically cleave JNK and NF-kappaB. The EMBO Journal.

NF-kappaB activation. PLoS Pathogens.

2009;**12**(1):101-109

2011;**30**(1):221-231

2010;**6**(1):e1000743

[56] Nadler C et al. The type III secretion effector NleE inhibits

[57] Sham HP et al. Attaching and effacing bacterial effector NleC suppresses epithelial inflammatory responses by inhibiting NF-{kappa} B and p38 mitogen-activated protein kinase activation. Infection and Immunity. 2011;**79**(9):3552-3562

[58] Gao X et al. NleB, a bacterial effector with glycosyltransferase activity, targets GADPH function to inhibit NF-κB activation. Cell Host &

Microbe. 2013;**13**(1):87-99

[59] Yen H, Sugimoto N, Tobe T. Enteropathogenic *Escherichia coli* uses NleA to inhibit NLRP3 inflammasome

activation. PLoS Pathogens.

2015;**11**(9):e1005121

mechanisms of *Escherichia coli* pathogenicity. Nature Reviews. Microbiology. 2010;**8**(1):26-38

2007;**2**(3):160-171

**94**

[71] Posfai G et al. Markerless gene replacement in *Escherichia coli* stimulated by a double-strand break in the chromosome. Nucleic Acids Research. 1999;**27**(22):4409-4415

[72] Hill SM, Phillips AD, Walker-Smith JA. Enteropathogenic *Escherichia coli* and life threatening chronic diarrhoea. Gut. 1991;**32**(2):154-158

[73] Deng W et al. Citrobacter rodentium translocated intimin receptor (Tir) is an essential virulence factor needed for actin condensation, intestinal colonization and colonic hyperplasia in mice. Molecular Microbiology. 2003;**48**(1):95-115

[74] Knutton S, Lloyd DR, McNeish AS. Adhesion of enteropathogenic *Escherichia coli* to human intestinal enterocytes and cultured human intestinal mucosa. Infection and Immunity. 1987;**55**(1):69-77

[75] Schüller S et al. Tir phosphorylation and Nck/N-WASP recruitment by enteropathogenic and enterohaemorrhagic *Escherichia coli* during ex vivo colonization of human intestinal mucosa is different to cell culture models. Cellular Microbiology. 2007;**9**(5):1352-1364

[76] McDaniel TK, Kaper JB. A cloned pathogenicity island from enteropathogenic *Escherichia coli* confers the attaching and effacing phenotype on *E. coli* K-12. Molecular Microbiology. 1997;**23**(2):399-407

[77] Riquelme-Neira R et al. Vaccination with DNA encoding truncated enterohemorrhagic *Escherichia coli* (EHEC) factor for adherence-1 gene

(efa-1′) confers protective immunity to mice infected with *E. coli* O157:H7. Frontiers in Cellular and Infection Microbiology. 2016;**5**:104-104

[78] Szu SC, Ahmed A. Clinical studies of *Escherichia coli* O157:H7 conjugate vaccines in adults and young children. Microbiology Spectrum. 2014;**2**(6):1-7

[79] Rabinovitz BC et al. The intranasal vaccination of pregnant dams with Intimin and EspB confers protection in neonatal mice from *Escherichia coli* (EHEC) O157:H7 infection. Vaccine. 2016;**34**(25):2793-2797

[80] Marcato P et al. Recombinant Shiga toxin B-subunit-keyhole limpet hemocyanin conjugate vaccine protects mice from Shigatoxemia. Infection and Immunity. 2005;**73**(10):6523-6529

[81] Piñero-Lambea C, Ruano-Gallego D, Fernández LÁ. Engineered bacteria as therapeutic agents. Current Opinion in Biotechnology. 2015;**35**:94-102

**97**

**Chapter 6**

**Abstract**

**1. Introduction**

the large intestine [5, 6].

infections in our environment [8, 9].

nal pathogenic lines [10].

**2. Epidemiology**

*and Nimisha Shiwalkar*

ing cause of neonatal meningitis.

Management of *E. coli* Sepsis

*Lais Sanches Maekawa, Roopvir Kaur, Silas Jose Braz Filo,* 

*E. coli* is the most common cause of urinary tract infections (UTIs) in humans and is a leading cause of enteric infections and systemic infections. The systemic infections include bacteremia, nosocomial pneumonia, cholecystitis, cholangitis, peritonitis, cellulitis, osteomyelitis, and infectious arthritis. *E. coli* is also the lead-

*Escherichia coli* strains compose, physiologically part of the microflora of the gastrointestinal tract [1–4]. Belonging to the Enterobacteriaceae family, fermentative, non-sporulated and facultative anaerobic commensals, they are mainly from

Despite being commensal microorganisms, they are the Gram-negatives which are most often a cause of human infections, having pathogenic strains that cause a wide variety of intestinal or extra-intestinal infections, such as urinary tract, intra-abdominal and soft tissue, sepsis, neonatal meningitis, gastrointestinal infection, and pneumonia, often leading to bacteremia [3, 7]. Although Gram-positive microorganisms have been increasing as a cause of sepsis due to the instrumentation of medical care—understood as the use of invasive devices or tools for the treatment or diagnosis of patients, and to infections associated with health care—*E. coli* continues to be an important and perhaps the most frequent cause of threatening

They are classified as Gram-negative bacteria and divided into 3 main groups: commensal lines, intestinal pathogenic lines (enteric or diarrhea) and extra-intesti-

Furthermore, Gram-negative bacteria produce large molecules consisting of a lipid and a polysaccharide, known as lipopolysaccharides (LPS), lipoglycans and endotoxin,

which increases their pathogenicity in relation to Gram-positive bacteria [11].

*E. coli* is one of the most commonly isolated bacteria in the bloodstream (responsible for approximately 20% of all clinically significant isolates) and is the

**Keywords:** bacteremia, septicemia, septic shock, antimicrobial therapy

*Purnadeo Persaud, Juber Dastagir Shaikh, Asim Kichloo* 

*Akshay Kumar, Ana Francesca Vommaro Leite,* 

#### **Chapter 6**

## Management of *E. coli* Sepsis

*Akshay Kumar, Ana Francesca Vommaro Leite, Lais Sanches Maekawa, Roopvir Kaur, Silas Jose Braz Filo, Purnadeo Persaud, Juber Dastagir Shaikh, Asim Kichloo and Nimisha Shiwalkar*

#### **Abstract**

*E. coli* is the most common cause of urinary tract infections (UTIs) in humans and is a leading cause of enteric infections and systemic infections. The systemic infections include bacteremia, nosocomial pneumonia, cholecystitis, cholangitis, peritonitis, cellulitis, osteomyelitis, and infectious arthritis. *E. coli* is also the leading cause of neonatal meningitis.

**Keywords:** bacteremia, septicemia, septic shock, antimicrobial therapy

#### **1. Introduction**

*Escherichia coli* strains compose, physiologically part of the microflora of the gastrointestinal tract [1–4]. Belonging to the Enterobacteriaceae family, fermentative, non-sporulated and facultative anaerobic commensals, they are mainly from the large intestine [5, 6].

Despite being commensal microorganisms, they are the Gram-negatives which are most often a cause of human infections, having pathogenic strains that cause a wide variety of intestinal or extra-intestinal infections, such as urinary tract, intra-abdominal and soft tissue, sepsis, neonatal meningitis, gastrointestinal infection, and pneumonia, often leading to bacteremia [3, 7]. Although Gram-positive microorganisms have been increasing as a cause of sepsis due to the instrumentation of medical care—understood as the use of invasive devices or tools for the treatment or diagnosis of patients, and to infections associated with health care—*E. coli* continues to be an important and perhaps the most frequent cause of threatening infections in our environment [8, 9].

They are classified as Gram-negative bacteria and divided into 3 main groups: commensal lines, intestinal pathogenic lines (enteric or diarrhea) and extra-intestinal pathogenic lines [10].

Furthermore, Gram-negative bacteria produce large molecules consisting of a lipid and a polysaccharide, known as lipopolysaccharides (LPS), lipoglycans and endotoxin, which increases their pathogenicity in relation to Gram-positive bacteria [11].

#### **2. Epidemiology**

*E. coli* is one of the most commonly isolated bacteria in the bloodstream (responsible for approximately 20% of all clinically significant isolates) and is the Gram-negative organism most frequently isolated in adult patients with bacteremia [12]. In the United States of America, *E. coli* sepsis was associated with approximately 40,000 deaths in 2001, a number that corresponds to 17% of all cases of sepsis [13].

Studies have shown an increasing incidence of *E. coli* early-onset sepsis in all age groups, overruling group B *Streptoccocus* for the last 10 years. Beyond that, *E. coli* resistant strains also increased equally in all age groups, with high resistance rates to first line antibiotics available (ampicillin and gentamicin).

Very low birth weight newborns remained the group with higher incidence (10.4 cases per 1000 live births) and mortality (35.3%). Systematic use of PCR increased *E. coli* early-onset sepsis diagnosis, mainly in the term newborn group. There was also an increase in resistant *E. coli* strains causing early-onset sepsis, with especially high resistance to ampicillin and gentamicin (92.8 and 28.6%, respectively) [14].

#### **3. Risk factors**

Several hospital-based studies have suggested that a number of comorbid illnesses, including diabetes, malignancy, chronic lung disease, cirrhosis and heart disease, may increase the risk of *E. coli* bacteremia. Previous researches have also identified age (very young and very elder), hospital acquisition, comorbid illnesses, presence of shock, non-urinary focus, and antimicrobial resistance in conjunction with inadequate treatment as being associated with higher rates of death [15–17].

Dialysis, solid organ transplantation and neoplastic disease were important risk factors for acquiring *E. coli* bacteraemia. Ciprofloxacin resistance and non-urinary focus were independently associated with an increased risk of death [18]. For males, urinary catheterization and incontinence were associated as risk factors to *Escherichia coli* bloodstream, and for females, cancer, renal failure, heart disease and urinary incontinence were risk factors reported [19]. Several risk factors which have significantly mortality due to *E. coli* bacteremia are age, severe sepsis or shock, non-urinary origin, Charlson index, inadequate empirical treatment (**Table 1**).


#### **Table 1.**

*Results of multivariate analyses examining risk factors for mortality associated with bacteraemia due to E. coli [15].*

#### **4. Pathogenesis**

The human gastrointestinal tract is normally inhabited by *Escherichia coli*, which is why they are the bacterial species most commonly found in the isolation

**99**

*Management of* E. coli *Sepsis*

dangerous) [22, 23].

**4.1 Intestinal**

toxins.

junctions [31].

*4.1.2 ETEC*

intestine [32].

*4.1.1 EPEC*

*DOI: http://dx.doi.org/10.5772/intechopen.93132*

of fecal culture [20, 21]. By the time the strains acquire additional genetic material, they can become pathogenic and circulate widely throughout the body. Pathological clones are divided into two major groups: intestinal (among the most virulent enteric pathogens) and extraintestinal (less present, but not less

Typical enteropathogenic *Escherichia coli* (tEPEC) contains a virulence plasmid (pEAF) that encodes the bundle-forming pilus (BFP), the primary factor for colonization [24, 25]. In addition, EPEC carries the crossomic island of locus for enterocyte effacement, which features the eae gene, which is the encoder of a colonization factor in the outer membrane protein called intimin [26, 27]. Only the *E. coli* strain that has pEAF and the eae gene can be considered tEPEC, one that has

The small intestine is the most likely place for EPEC infection to occur. For the

• Induction of signal transduction in the enterocyte by secretion of protein

• Development of intimin-mediated intimate adhesion to the enterocyte.

Around 20 protein toxins are injected directly into the target epithelial cell, made, together with the intimin, by the chromosomal island LEE and expressed by both tEPEC and aEPEC [29]. The complex nanomachine called type III secretion injector is the one that injects protein toxins. It is assumed that some modifications happen to the epithelial stem cells, which is physiologically absorbent, and through

What is believed is that type III ejection toxins are responsible for binding to protein elements of the cell's signal transduction apparatus. This event is accompanied by the mobilization of calcium from the intracellular compartment, activation of protein kinase C, kinase light chain myosin and induction of protein phosphorylation by tyrosine. The rearrangement of cytoskeletal proteins is induced by effectors, which results in the classic lesion "attaching and erasing," changes in the secretion of water and electrolytes and increased permeability of the tight intestinal

Enterotoxigenic *Escherichia coli* (ETEC) consists of ingestion of bacteria, intestinal colonization and production of virulence factors. Colonizing fimbriae (CFs) must be expressed by ETEC to allow the consolidation of the bacteria in the

After colonization, ETEC produces two classes of secretory toxins encoded by plasmids: heat-labile toxin (LT) and heat-stable toxin (ST). To be classified as

LT toxin is related to Vibrio cholera toxins in terms of structure, function and mechanism. It works by stimulating adenylate cyclase and increasing adenosine

ETEC, *E. coli* must contain one or both classes of toxins [33, 34].

only the eae gene and is called atypical EPEC (aETEC) [28].

a pathological process, it becomes a secretory dynamo [30].

• Initial localized adhesion of organisms to enterocyte via BFP.

onset of diseases, tEPEC obeys the following steps:

#### *Management of* E. coli *Sepsis DOI: http://dx.doi.org/10.5772/intechopen.93132*

of fecal culture [20, 21]. By the time the strains acquire additional genetic material, they can become pathogenic and circulate widely throughout the body. Pathological clones are divided into two major groups: intestinal (among the most virulent enteric pathogens) and extraintestinal (less present, but not less dangerous) [22, 23].

#### **4.1 Intestinal**

#### *4.1.1 EPEC*

E. Coli *Infections - Importance of Early Diagnosis and Efficient Treatment*

first line antibiotics available (ampicillin and gentamicin).

sepsis [13].

respectively) [14].

**3. Risk factors**

death [15–17].

(**Table 1**).

Gram-negative organism most frequently isolated in adult patients with bacteremia [12]. In the United States of America, *E. coli* sepsis was associated with approximately 40,000 deaths in 2001, a number that corresponds to 17% of all cases of

Studies have shown an increasing incidence of *E. coli* early-onset sepsis in all age groups, overruling group B *Streptoccocus* for the last 10 years. Beyond that, *E. coli* resistant strains also increased equally in all age groups, with high resistance rates to

Very low birth weight newborns remained the group with higher incidence (10.4 cases per 1000 live births) and mortality (35.3%). Systematic use of PCR increased *E. coli* early-onset sepsis diagnosis, mainly in the term newborn group. There was also an increase in resistant *E. coli* strains causing early-onset sepsis, with especially high resistance to ampicillin and gentamicin (92.8 and 28.6%,

Several hospital-based studies have suggested that a number of comorbid illnesses, including diabetes, malignancy, chronic lung disease, cirrhosis and heart disease, may increase the risk of *E. coli* bacteremia. Previous researches have also identified age (very young and very elder), hospital acquisition, comorbid illnesses, presence of shock, non-urinary focus, and antimicrobial resistance in conjunction with inadequate treatment as being associated with higher rates of

Dialysis, solid organ transplantation and neoplastic disease were important risk factors for acquiring *E. coli* bacteraemia. Ciprofloxacin resistance and non-urinary focus were independently associated with an increased risk of death [18]. For males, urinary catheterization and incontinence were associated as risk factors to *Escherichia coli* bloodstream, and for females, cancer, renal failure, heart disease and urinary incontinence were risk factors reported [19]. Several risk factors which have significantly mortality due to *E. coli* bacteremia are age, severe sepsis or shock, non-urinary origin, Charlson index, inadequate empirical treatment

The human gastrointestinal tract is normally inhabited by *Escherichia coli*, which is why they are the bacterial species most commonly found in the isolation

*Results of multivariate analyses examining risk factors for mortality associated with bacteraemia due to* 

**Mortality risk factor** *P* **OR (95% CI)** Age 0.03 1.04 (1–1.08) Severe sepsis or shock <0.0001 14.64 (6.14–30.86) Non-urinary origin 0.013 2.78 (1.24–6.2) Charlson index 0.006 1.31 (1.08–1.59) Inadequate empirical treatment 0.006 2.98 (1.25–7.11)

**98**

**Table 1.**

*E. coli [15].*

**4. Pathogenesis**

Typical enteropathogenic *Escherichia coli* (tEPEC) contains a virulence plasmid (pEAF) that encodes the bundle-forming pilus (BFP), the primary factor for colonization [24, 25]. In addition, EPEC carries the crossomic island of locus for enterocyte effacement, which features the eae gene, which is the encoder of a colonization factor in the outer membrane protein called intimin [26, 27]. Only the *E. coli* strain that has pEAF and the eae gene can be considered tEPEC, one that has only the eae gene and is called atypical EPEC (aETEC) [28].

The small intestine is the most likely place for EPEC infection to occur. For the onset of diseases, tEPEC obeys the following steps:


Around 20 protein toxins are injected directly into the target epithelial cell, made, together with the intimin, by the chromosomal island LEE and expressed by both tEPEC and aEPEC [29]. The complex nanomachine called type III secretion injector is the one that injects protein toxins. It is assumed that some modifications happen to the epithelial stem cells, which is physiologically absorbent, and through a pathological process, it becomes a secretory dynamo [30].

What is believed is that type III ejection toxins are responsible for binding to protein elements of the cell's signal transduction apparatus. This event is accompanied by the mobilization of calcium from the intracellular compartment, activation of protein kinase C, kinase light chain myosin and induction of protein phosphorylation by tyrosine. The rearrangement of cytoskeletal proteins is induced by effectors, which results in the classic lesion "attaching and erasing," changes in the secretion of water and electrolytes and increased permeability of the tight intestinal junctions [31].

#### *4.1.2 ETEC*

Enterotoxigenic *Escherichia coli* (ETEC) consists of ingestion of bacteria, intestinal colonization and production of virulence factors. Colonizing fimbriae (CFs) must be expressed by ETEC to allow the consolidation of the bacteria in the intestine [32].

After colonization, ETEC produces two classes of secretory toxins encoded by plasmids: heat-labile toxin (LT) and heat-stable toxin (ST). To be classified as ETEC, *E. coli* must contain one or both classes of toxins [33, 34].

LT toxin is related to Vibrio cholera toxins in terms of structure, function and mechanism. It works by stimulating adenylate cyclase and increasing adenosine

intracellular cyclic monophosphate (AMP), a fact that stimulates chloride secretion from intestinal crypt cells and inhibits the absorption of sodium chloride at the ends of the villi. After that, the water secretion is free in the intestinal lumen, clinically developing watery diarrhea [35].

STa toxin, the only ST variant that causes disease in humans, activates cyclic GMP of enterocytes, leading to increased chloride secretion and decreased sodium chloride absorption. As a final result, the secretion of free water in the intestinal lumen clinically appears as watery diarrhea [36].

#### *4.1.3 EHEC*

Among the pathotypes that cause the most severe conditions, the strains classified as enterohemorrhagic (EHEC) stand out, which are the most common to cause disease in developed countries [29].

They are bacteria responsible for food infections and represent a risk to the health of the population, so they must be monitored frequently. Thus, good hygiene practices, as well as the use of quality tools, are extremely important to help reduce the risk of cross-contamination and human infection.

EHEC has the ability to attach itself to the host and to produce shiga-toxins, which gives the strain pathogenicity. The toxins produced by EHEC cause damage to the mucosa of the large intestine, where they are absorbed by reaching the bloodstream, which makes it possible to affect other organs, such as the kidneys [37]. An average of 5–10% of patients confirmed with EHEC infection develop potentially fatal complications, such as hemolytic uremic syndrome (HUS), which leads to sudden renal failure and hemolytic anemia [38].

Outbreaks are related to the ingestion of contaminated food and water, causing watery diarrhea and hemorrhagic colitis to those infected. The disease has a sudden onset with severe abdominal cramps and watery diarrhea that progresses to bloody, on average after 24 hours, lasting between 1 to 8 days.

The treatment consists of supportive therapy for fluid replacement, since the use of antibiotics is not indicated, as there is no proven efficacy. In fact, it could increase the risk of developing HUS, since the death of the bacteria would increase the release of toxins, predisposing to the syndrome [39].

#### *4.1.4 EIEC*

Enteroinvasive *E. coli* (EIEC) is very close to Shigella and develops a colitis similar to shigellosis. The intestinal cell is invaded by the EIEC which multiplies intracellularly and reaches the adjacent intestinal cells [40].

To differentiate Shigella from EIEC it is necessary to analyze the strains, those from EIEC ferment glucose and xylose, this differentiates them. Nucleic acid tests, including multiplexed panels, are used to detect organisms [41].

#### *4.1.5 DAEC*

Diffusely adherent *E. coli* is associated with diarrhea, which is characterized as watery and can become persistent in children between 1 and 5 years of age, occurring more frequently in developing and developed countries. In addition, this bacterium is also related to urinary tract infections and complications during the pregnancy period.

The pattern of diffuse adhesion in HEp-2 or HeLa cells is a characteristic that differentiates this pathotype from the others, although DAEC strains are quite

**101**

*Management of* E. coli *Sepsis*

atypical DAEC [42].

**4.2 Extraintestinal**

of foodborne pathogens [45].

*4.2.1 Urosepsis*

*DOI: http://dx.doi.org/10.5772/intechopen.93132*

in terms of medical costs and lost productivity [44].

antimicrobial resistance, and patient comorbidity.

products being more controversial in the literature [52].

"threatens the achievements of modern medicine" [53, 54].

temporal trends for antibiotic resistance to *E. coli*.

**5. Antimicrobial resistance**

heterogeneous. This adhesion is mediated by fimbrial and afimbrial adhesins, which can cause damage to microvilli due to the disorganization of the cytoskeleton. However, some strains produce an adhesin involved in diffuse adhesion (AIDA-I), instead of encoding the diffuse adhesion pattern, which is why they are called

In addition, DAEC can also provide a pro-inflammatory effect [43].

The type of *E. coli* responsible for the invasion, colonization and induction of diseases in body sites outside the gastrointestinal tract is the extraintestinal pathogenic *Escherichia coli* (ExPEC). It is noteworthy that diseases caused by ExPEC range from urinary tract infections, neonatal meningitis, sepsis, pneumonia, surgical site infections to infections in other extraintestinal sites, representing a burden

Thereto, the ExPEC strains were isolated from food products, in particular raw meat and poultry, indicating that these organisms potentially represent a new class

Almost 25% of sepsis cases originate from the urogenital tract. [46–48]. Considering this percentage, the most common pathogen that causes urinary tract infection (and, consequently, urosepsis) is *Escherichia coli* (50%) [49]. It is known that this condition is better managed with an interprofessional team of health professionals—a nephrologist, infectious disease expert, urologist, intensivist, a nurse and a pharmacist [50, 51]. The outcomes after urosepsis depend on the cause and severity of the infection, and if the patient has a complicating factor in the urinary tract that is identified and warrants treatment, it should be performed as soon as possible. As an example, the literature reveals Foley catheter placement to relieve urinary retention or stent placement to bypass an obstructing ureteral calculus causing urosepsis. Moreover, the prognosis also depends on the type of bacteria,

In addition to early antibiotics, there are some important parts of the management of sepsis. Initial fluid resuscitation with crystalloid is still recommended at a minimum of 30 mL/kg. Consider early administration of vasopressor support to maintain a mean arterial pressure greater than 65 mm Hg. The first choice for vasopressor support in sepsis is norepinephrine (with epinephrine and vasopressin 2 and 3). Tight glucose control is also recommended, with corticosteroids and blood

Although *Escherichia coli* is one of the most-studied microorganisms worldwide, its characteristics are constantly changing. Elseways, one important global problem is the increase of antimicrobial resistance shown by bacteria, being considered as

*E. coli* resistant strains increased equally in all age groups, with high resistance rates to our first line antibiotics (ampicillin and gentamicin), with relevant highlight in neonatal *E. coli* isolates from invasive infection [55]. **Table 2** shows the

#### *Management of* E. coli *Sepsis DOI: http://dx.doi.org/10.5772/intechopen.93132*

heterogeneous. This adhesion is mediated by fimbrial and afimbrial adhesins, which can cause damage to microvilli due to the disorganization of the cytoskeleton. However, some strains produce an adhesin involved in diffuse adhesion (AIDA-I), instead of encoding the diffuse adhesion pattern, which is why they are called atypical DAEC [42].

In addition, DAEC can also provide a pro-inflammatory effect [43].

#### **4.2 Extraintestinal**

E. Coli *Infections - Importance of Early Diagnosis and Efficient Treatment*

clinically developing watery diarrhea [35].

disease in developed countries [29].

*4.1.3 EHEC*

*4.1.4 EIEC*

*4.1.5 DAEC*

pregnancy period.

lumen clinically appears as watery diarrhea [36].

the risk of cross-contamination and human infection.

sudden renal failure and hemolytic anemia [38].

on average after 24 hours, lasting between 1 to 8 days.

the release of toxins, predisposing to the syndrome [39].

intracellularly and reaches the adjacent intestinal cells [40].

including multiplexed panels, are used to detect organisms [41].

intracellular cyclic monophosphate (AMP), a fact that stimulates chloride secretion from intestinal crypt cells and inhibits the absorption of sodium chloride at the ends of the villi. After that, the water secretion is free in the intestinal lumen,

STa toxin, the only ST variant that causes disease in humans, activates cyclic GMP of enterocytes, leading to increased chloride secretion and decreased sodium chloride absorption. As a final result, the secretion of free water in the intestinal

Among the pathotypes that cause the most severe conditions, the strains classified as enterohemorrhagic (EHEC) stand out, which are the most common to cause

They are bacteria responsible for food infections and represent a risk to the health of the population, so they must be monitored frequently. Thus, good hygiene practices, as well as the use of quality tools, are extremely important to help reduce

EHEC has the ability to attach itself to the host and to produce shiga-toxins, which gives the strain pathogenicity. The toxins produced by EHEC cause damage to the mucosa of the large intestine, where they are absorbed by reaching the bloodstream, which makes it possible to affect other organs, such as the kidneys [37]. An average of 5–10% of patients confirmed with EHEC infection develop potentially fatal complications, such as hemolytic uremic syndrome (HUS), which leads to

Outbreaks are related to the ingestion of contaminated food and water, causing watery diarrhea and hemorrhagic colitis to those infected. The disease has a sudden onset with severe abdominal cramps and watery diarrhea that progresses to bloody,

The treatment consists of supportive therapy for fluid replacement, since the use of antibiotics is not indicated, as there is no proven efficacy. In fact, it could increase the risk of developing HUS, since the death of the bacteria would increase

Enteroinvasive *E. coli* (EIEC) is very close to Shigella and develops a colitis similar to shigellosis. The intestinal cell is invaded by the EIEC which multiplies

To differentiate Shigella from EIEC it is necessary to analyze the strains, those from EIEC ferment glucose and xylose, this differentiates them. Nucleic acid tests,

Diffusely adherent *E. coli* is associated with diarrhea, which is characterized as watery and can become persistent in children between 1 and 5 years of age, occurring more frequently in developing and developed countries. In addition, this bacterium is also related to urinary tract infections and complications during the

The pattern of diffuse adhesion in HEp-2 or HeLa cells is a characteristic that differentiates this pathotype from the others, although DAEC strains are quite

**100**

The type of *E. coli* responsible for the invasion, colonization and induction of diseases in body sites outside the gastrointestinal tract is the extraintestinal pathogenic *Escherichia coli* (ExPEC). It is noteworthy that diseases caused by ExPEC range from urinary tract infections, neonatal meningitis, sepsis, pneumonia, surgical site infections to infections in other extraintestinal sites, representing a burden in terms of medical costs and lost productivity [44].

Thereto, the ExPEC strains were isolated from food products, in particular raw meat and poultry, indicating that these organisms potentially represent a new class of foodborne pathogens [45].

#### *4.2.1 Urosepsis*

Almost 25% of sepsis cases originate from the urogenital tract. [46–48]. Considering this percentage, the most common pathogen that causes urinary tract infection (and, consequently, urosepsis) is *Escherichia coli* (50%) [49]. It is known that this condition is better managed with an interprofessional team of health professionals—a nephrologist, infectious disease expert, urologist, intensivist, a nurse and a pharmacist [50, 51]. The outcomes after urosepsis depend on the cause and severity of the infection, and if the patient has a complicating factor in the urinary tract that is identified and warrants treatment, it should be performed as soon as possible. As an example, the literature reveals Foley catheter placement to relieve urinary retention or stent placement to bypass an obstructing ureteral calculus causing urosepsis. Moreover, the prognosis also depends on the type of bacteria, antimicrobial resistance, and patient comorbidity.

In addition to early antibiotics, there are some important parts of the management of sepsis. Initial fluid resuscitation with crystalloid is still recommended at a minimum of 30 mL/kg. Consider early administration of vasopressor support to maintain a mean arterial pressure greater than 65 mm Hg. The first choice for vasopressor support in sepsis is norepinephrine (with epinephrine and vasopressin 2 and 3). Tight glucose control is also recommended, with corticosteroids and blood products being more controversial in the literature [52].

#### **5. Antimicrobial resistance**

Although *Escherichia coli* is one of the most-studied microorganisms worldwide, its characteristics are constantly changing. Elseways, one important global problem is the increase of antimicrobial resistance shown by bacteria, being considered as "threatens the achievements of modern medicine" [53, 54].

*E. coli* resistant strains increased equally in all age groups, with high resistance rates to our first line antibiotics (ampicillin and gentamicin), with relevant highlight in neonatal *E. coli* isolates from invasive infection [55]. **Table 2** shows the temporal trends for antibiotic resistance to *E. coli*.

