## 3 Mode of Action of the ClyA Toxin as a Virulence Factor  
ClyA is a widely known PFT. Unlike _E. coli_ HlyA and EhxA, which are posttranslationally modified by the addition of fatty acids [59; 60; 61], ClyA does not require posttranslational modification to be active. However, ClyA activity is tightly controlled at the transcriptional level under certain conditions and by regulation systems involving several regulators, as described above (see Section 2.2), and is also influenced by structural changes or environmental signals. As described in Section 2.3, ClyA oligomerization is essential for full activity. For example, the structural change from the monomeric to the oligomeric form is crucial for ClyA activity and is involved in the altered redox status [20]. However, another study has shown that the intrinsic hemolytic activity of ClyA is independent of its redox state, and that assembly of both reduced and oxidized ClyA into the ring-shaped oligomer is triggered by contact with membrane lipids or a detergent [55]. ClyA harbors a cholesterol recognition and consensus (CRAC) motif, which typically stabilizes structural intermediates in assembly pathways that occur in the presence of cholesterol. The ClyA region containing this CRAC motif, which comprises residues L24-Y27-K29 (\(N\)-terminal helix), is conserved in ClyA homologs from several bacterial species (_E. coli_, _S._ Typhi, _S._ Paratyphi, _Bacillus subtilis_, and _S. sonnei_), and the presence of cholesterol stimulates pore formation by selectively stabilizing a protomer-like conformation, leading to hemolysis [62]. Figure 1: Schematic representation of the OMV-mediated secretion pathway for ClyA and the predicted pore assembly pathway. ClyA protein is secreted by OMVs and the monomer oligomerizes to form the active pore assemblies within OMVs in a redox state-dependent manner [20]. In the assembly process, once ClyA reaches the target membrane, the monomer first undergoes a conformational change to a protomer and then predominantly assembles into an octameric [53], 13-meric (redox state-independent) [55], or dodecameric [14] pore complex by contact with membrane lipids or the detergent, \(n\)-dodecylmaloside (DDM). ClyA-mediated cell lysis involves a complex series of steps in which ClyA must recognize and bind to the target cell and then assemble to form a functional pore. A complete preproc is assembled on the target membrane (or, in the non-classical pathway, a soluble preproc forms within the OMVs); the membrane then becomes distorted, and a functional pore is formed by the insertion of \(\alpha\)-helices into the lipid bilayer, resulting in hemolysis-like cell lysis [14; 16; 20; 52; 53]. In addition to hemolysis, it has been reported that ClyA has other cytotoxic effects on target cells. [PERSON] et al. demonstrated experimentally that highly purified ClyA exhibited cytotoxic activity toward J774 murine macrophage-like cells, which was associated with morphology changes and a substantial decrease in the detachment of target cells [63]. They speculated that membrane cholesterol may stimulate ClyA lytic action, and this effect was investigated in a subsequent study conducted by [PERSON] et al. [62]. Another group also demonstrated that purified ClyA and a ClyA-expressing _E. coli_ strain were cytotoxic to both human and murine macrophages, and that this cytotoxicity was dose and time dependent. Their findings also suggested that ClyA induces massive apoptosis, as indicated by host-cell DNA fragmentation [64]. Thus, the pore-forming activity of ClyA is responsible for the induction of apoptosis in target cells, similar to the pore-forming _S. aureus_\(\alpha\)-toxin and _E. coli_ HlyA [65; 66]. In addition, [PERSON] and coworkers showed that ClyA assisted _S._ Typhi to invade human epithelial cells in vitro and promoted the colonization of deep organs in mice when expressed heterologously in _S._ Typhimurium [67]. Toxins can have different biological effects on target cells, depending on the concentration.