*3.2.1 Colonization and adherence*

Ingestion of contaminated food or direct contact with contaminated environmental matrices or infected persons or animals precedes STEC-related diseases. Colonization and attachment/adherence of STEC to intestinal epithelium is mediated by several genetic factors some of which are carried by the locus of enterocyte effacement (LEE) PAI [65]. LEE locus which is also often present in EPEC strains [66] encodes a type III secretion system (T3SS) that plays a role in the secretion and translocation of virulence-associated genetic factors into host cells [67]. One of these genetic factors is *eae* gene that encodes intimin, an adhesin that is essential for the attachment of STEC to the host intestinal mucous membrane and facilitates the production of attaching-and-effacing (A/E) lesion (**Figure 3**) [65]. The injection of its translocated intimin receptor (Tir) into the host cells and the interaction between this protein (Tir), proteins that form the needle component of the T3SS (EspADB), and intimin facilitates the induction of lesions [65, 68]. Intimin has at least 30 reported subtypes [69] and some of these variants are associated with specific serotypes. Oftentimes, intimin encoding gene (*eae*) subtype γ1 is associated with STEC O157:H7 and O145:H28 [69, 70], while subtypes β1, ε, and θ are frequently found in O26:H11, O103:H2 and O111:H8 STEC strains, respectively [69, 70]. Intimin is reported to be common in clinical strains of STEC with a prevalence that could range from 70 to 90% [71–73].

LEE is unarguably important for the pathogenesis of STEC strains, but STEC LEEnegative strains/serotypes (e.g. O103:H21) have been implicated in infections [74].

This implies that there are several other virulence-associated factors that are carried on PAIs or mobile genetic elements mediating colonization and adherence to host cells in these strains [74]. For example, a gene (*saa*) encoding autoagglutinating adhesin was isolated from a large plasmid of a LEE-negative STEC O113:H21 implicated in an outbreak. The expression of this gene is described to enhance adherence of STEC strains to HEp-2 cells [75]. Other protein-encoding genes reported to promote the colonization and adherence of STEC strains to mucosal membrane include *paa* that encodes attachment of bacterial cells to enterocytes in pigs [76], *ehaA* a STEC autotransporter [77], *lpfA,* long polar fimbriae [78] that are both involved in the attachment of STEC to surfaces. Additionally, "O" islands (OI) that encode macrophage toxin and ClpB-like chaperone (OI-7), urease clusters (OI-43 and OI-48), two toxins and PagC-like virulence factor (OI-122) among others have been reported to be linked with virulence in STEC [57, 79].

#### *3.2.2 Cytotoxic toxin production*

STEC-related infections in humans are reported to be associated with the presence and expression of several virulence determinants, with the phage-encoded Shiga toxin genes Stx1 (Stx1a) and Stx2 (Stx2a) being the main virulence factors [64, 80]. Stx1 consists of 293 amino acids while the Stx2a is longer by only four amino acids. At least, 16 subtypes of these two toxins have been described based on amino acid differences and the level of cytotoxicity. Stx1 contains four variants encoded by *stx1a*, *stx1c*, *stx1d* and *stx1e*, whereas Stx2 comprised 12 subtypes encoded by *stx2a*, *stx2b*, *stx2c*, *stx2d*, *stx2e*, *stx2f*, *stx2g, stx2h, stx2i, stx2j, stx2k* and *stx2l* [80]. While Stx1a has been implicated in human infection, Stx2a, Stx2c, and Stx2d are the major subtypes that are frequently associated with hemorrhagic colitis and HUS [64]. However, Stx2a and Stx2d subtypes are described to exhibit higher cytotoxicity relative to Stx2b and Stx2c in a mouse model [64, 81]. The interaction of Stxs with the host cell receptor is very complex and is based on characteristics of the environment of the receptor in the plasma membrane [64]. Stxs bind to the globotriaosylceramide Gb3, an insoluble molecule that has multiple binding sites and comprised a lipid component. The interaction between these two molecules (Stx and Gb3) is described to be important in the uptake of the toxin (**Figure 3**). Stxs are ribotoxins that disrupt protein synthesis within the host cell and provoke apoptosis [64, 81].

STEC strains can carry *stx1* or *stx2* genes or both [2]. In a study of 351 STEC strains from bovine feces, the great majority of the strains (82%) carried *stx2* while 18% carried *stx*1. Both genes were found only in ~3% of the collection [2]. In another study involving 220 STEC strains from humans and animals, *stx1* and *stx2* were found in 15% and 53%, respectively, while both genes were found in 32% of the isolates [82]. Stxs subtypes are heterogeneously distributed in the population, but specific variants have been reported to be host-specific. For example, Stx2e is less cytotoxic and sporadic in human diseases, and is commonly associated with edema diseases in weaned pigs [83]. Likewise, Stx1c is reported to be associated with STEC of ovine origin [84]. Indeed, the severity of STEC infection has been noted to be directly proportional to a number of Stx types or subtypes carried by the infecting strains [85]. While the production of only Stxs has been described to cause HUS, the infection is however exacerbated when associated with other virulence determinants including the LEE [21]. In addition to Stx, other toxins or hemolysins have been reported to be associated with STEC virulence. These include the hemolysin, encoded by the *ehxA* or *hlyA* gene, that are usually found on megaplasmid pO113 and/or pO157 and linked to cytotoxic effects on endothelial cells that may also promote the development of HUS [86, 87]. Other virulence

determinants carried on these plasmids include *toxB* that is essential for adherence of STEC to host cells [88], *espP* that encodes an extracellular protease and *katP* that is associated with catalase-peroxidase production important for oxidative stress response [89].
