STEC’s characteristic virulence factors are located on the bacterial chromosome and on
plasmids. Important virulence genes of STEC are (i) the attachment and effacement gene eae, (ii) the ehxA gene, and (iii) the Shiga toxin genes stx1 and stx2, which play a crucial role in the pathogenesis of STEC infection in the host. Table 2.2 summarises the main virulence factors of STEC, their genomic location in the bacterium, microbial structures and functions.
Table 2.2: A summary of main virulence factors of STEC including their genomic location, bacterial structure, and functions in the pathogenesis of STEC infections.
Virulence factor Gene
Genomic location Microbial structure Function in STEC pathogenesis Intimin eae Chromosome,
LEEa Outer membrane protein Mediating intimate attachment of STEC to enterocytes
EspA espA Chromosome, LEE
Secretory protein
Forming needle-like structure for injection of EspB, EspC, EspD, and Tir into enterocytes (part of T3SSb)
EspB, EspC, EspD espB,
espC, espD Chromosome, LEE Secretory proteins (part of T3SS)
Tir tir Chromosome,
LEE
Secretory protein
Receptor for intimin in cell membrane of enterocytes (part of T3SS)
Entero- haemolysin
ehxA Plasmid (pO157) Structural protein
Still unknown Serine protease
(EspP)
espP Plasmid (pO157) Enzyme Probably contributing to mucosal haemorrhage Catalase-
peroxidase (KatP)
katP Plasmid (pO157) Enzyme ? T2SSc etp Plasmid (pO157) Secretory
protein
?
Shiga toxin (Stx) stx Bacteriophage Toxin Inhibiting enterocyte’s protein synthesis, exact role still not fully understood
a
LEE = Locus for enterocyte effacement.
b
T3SS = Type III secretion system.
C
Attachment and effacement gene (eae)
The attachment and effacement gene eae and other important virulence genes are responsible for the formation of attachment and effacement (A/E) lesions on intestinal epithelial cells. Characteristics of A/E lesions are the intimate attachment of STEC to enterocytes, the effacement of microvilli and pedestal-like formation of enterocytes [43]. The eae gene encodes a bacterial outer membrane protein, intimin, which mediates the intimate attachment of STEC to the enterocytes [43]. It is located on a large chromosomal pathogenicity island
known as the ‘locus for enterocyte effacement’ (LEE) [122], which harbours also other important virulence genes including espA, espB, espC, espD (encoding for E. coli secretory proteins EspA to EspD) and tir (encoding for translocation intimin receptor Tir) [123], which are part of the type III secretion system (T3SS). EspA forms a filamentous structure through which EspB, EspC, EspD, and Tir are injected into the cytoplasm of the enterocyte [124]; Tir is then inserted as a receptor for intimin in the cell membrane of the enterocyte [123]. The typical A/E lesions are formed when intimin attaches to Tir [125].
Enterohaemolysin gene (ehxA)
The enterohaemolysin gene ehxA, also known as hlyA, is an important virulence marker of STEC and has been associated with clinical disease in humans; however its precise role in the complex mechanism of STEC pathogenesis is still not fully understood. ehxA encodes for the structural enterohaemolysin protein, which causes haemolysis of washed sheep erythrocytes [126-128]. In vitro studies have indicated that enterohemolysin from an STEC O128:H12 isolate increased the levels of proinflammatory cytokine interleukin-1β from human monocytes [126]. The ehxA gene is located on a plasmid, an extrachromosomal DNA structure (92 kb to 104 kb) capable of replicating independently from the DNA chromosome. Toth et al. [129] named this plasmid pO157, which is carried by almost all E. coli O157:H7 strains. Enterohaemolysin was the first described virulence factor of the pO157 plasmid [130, 131]. In addition to ehxA, pO157 also harbours other virulence factors involved in the pathogenesis of STEC including serine protease (espP) [132], catalase-peroxidase (katP) [133], and type II secretion system (T2SS) (etp) [134].
Shiga toxin genes (stx)
stx is the most important virulence marker of STEC. STEC can produce two types of Shiga toxins (Stx) referred to as Stx1 and Stx2. The toxins are encoded on two separate stx genes (stx1 and stx2), which are associated with separate bacteriophage and acquired by the bacterial host through transduction [57, 135]. An STEC strain can possess the stx1 or stx2 gene, or both genes expressing Stx. Stx1 and Stx2 are serologically different Stxs, showing a homology of only 58% and 56% at the amino acid and nucleotide levels, respectively [135, 136]. Allelic variants have been described for both types of Stx, with three genetic subtypes for Stx1 (Stx1, Stx1c, and Stx1d) and more than 10 subtypes for Stx2, including Stx2, Stx2a Stx2c, Stx2d, Stx2e, Stx2f, and Stx2g [137]. Stx2 is more frequently associated with haemorrhagic colitis and HUS in clinical cases compared to Stx1 [138, 139]. Furthermore, data suggest that clinical manifestations of STEC infection in humans depend on the stx genetic subtype of the infecting strain. For example, Friedrich et al. [140] observed a significant association between genetic variants stx2d and stx2e (gene variants of stx encoding for Stx2d and Stx2e) and diarrhoea, while stx2c was the only variant associated with HUS. Stxs contain a structure consisting of one A subunit (A1) and five identical receptor-binding B subunits (B1 to B5). To enter the enterocyte, B5 subunit binds the toxin to a specific receptor (globotriaosylceramide or Gb3) on the surface of the enterocyte [43]. After binding to the
cell, Stx (A1B5) is endocytosed and the A subunit is translocated through the cytoplasm to the Golgi apparatus and endoplasmic reticulum to inhibit the cell’s protein synthesis [141]. After uptake and translocation of Stxs by enterocytes, Stxs are distributed systemically and bind to susceptible cells in other organs, i.e. kidneys, where Gb3 receptors are present at high
concentrations [142]. The exact role of Shiga toxins in mediating intestinal disease, HUS, and neurological disorder is still not fully understood.