## 2 General Features of ClyA
### Secretion Pathway  
Bacterial toxin transport across bacterial membranes to reach their targets is mediated by a variety of cotranslational and posttranslational modifications. Toxin transport occurs \begin{table}
\begin{tabular}{c c c c c} \hline \hline
**Gene** & **Product/Function** & **Organisms** & **Relevant Descriptions** & **References** \\ \hline _hns_ & heat-stable & _E. coli_ & strongly repress the transcriptional expression of _clyA_ gene under laboratory condition & [12; 13; 28; 29; 39] \\ \hline _slyA_ & MarR-family & _E. coli_ & activate the expression of _clyA_ gene & \\  & transcriptional regulator & _S._ Typhimuriumur & by antagonizing the H-NS-mediated transcriptional repression when & [12; 13; 28; 29; 39] \\  & & & & overexpressing SlyA & \\ \hline _mprA_ & MarR-family & _E. coli_ & activate the expression of _clyA_ gene & \\  & transcriptional regulator & _E. coli_ & by antagonizing the H-NS-mediated transcriptional repression when & [24] \\  & & & & overexpressing MprA & \\ _fur_ & fumarate and nitrate & _E. coli_ & activate the expression of _clyA_ gene & \\  & reduction regulator & _E. coli_ & in response to oxygen depletion & [33; 35] \\ \hline _crp_ & cyclic AMP receptor protein & _E. coli_ & activate the expression of _clyA_ gene & \\  & & & & in response to glucose starvation & \\  & cyclic AMP receptor protein & _S._ Typhi & repress the transcriptional expression of _clyA_ gene (on SPI-18) & [37] \\  & & & & via down-regulating _rpoS_ & \\ \hline \multirow{2}{*}{_phoP_} & transcriptional regulator & \multirow{2}{*}{_S._ Typhi} & up-regulate the expression of _clyA_ & \\  & (two-component regulatory & & gene (on SPI-18) via _rpoS_ under low & [36; 38] \\  & system PhoP/PhoQ) & & pH and low Mg\({}^{2+}\) & \\ \hline \multirow{2}{*}{_fis_} & \multirow{2}{*}{DNA-binding protein} & \multirow{2}{*}{_S._ Typhi} & down-regulate the expression of & \\  & & & _clyA_ gene (on SPI-18) in & \\  & & & CRP-independent & \\  & & & glucose-dependent manner & \\ \hline \multirow{4}{*}{_rpoS_} & \multirow{4}{*}{RNA polymerase sigma factor} & \multirow{4}{*}{_S._ Typhi} & relate to the transcriptional & \\  & & & upregulation of _clyA_ gene (on & \\ \cline{1-1}  & & & SPI-18) in low pH and high & \\ \cline{1-1}  & & & osmolarity condition (predicted & \\ \cline{1-1}  & & & central regulator in the _clyA_ & \\ \cline{1-1}  & & & regulatory network) & \\ \hline \hline \end{tabular}
\end{table} 
Table 1: List of major transcriptional regulators for _clyA_ gene expression. by multiple mechanisms, which have been characterized in both Gram-negative and Gram-positive bacteria [1]. For example, the _E. coli_ hemolysis HlyA has a signal sequence at the \(C\)-terminus and is secreted by a type 1 secretion system (T1 SS) in a single step directly from the cytosol to the extracellular space [40; 41]. However, ClyA lacks a canonical signal sequence and undergoes no \(N\)-terminal processing during secretion [24; 39]. In addition, substantial amounts of periplasmic proteins have been detected in culture supernatants from ClyA-overproducing cells [39], implying that ClyA secretion is accompanied by leakage of periplasmic contents. These findings indicated that ClyA is not secreted by general secretion machinery, such as a T1 SS system, raising the question of how ClyA reaches the extracellular space. In addition to known secretion systems, most bacteria also utilize membrane vesicle-mediated transport to secrete various toxins and deliver them to target cells. Bacterial membrane vesicles are spherical blebs with an average diameter of 20-300 nm that are naturally released from both Gram-negative and Gram-positive bacteria, and are often referred to as outer membrane vesicles (OMVs), especially in Gram-negative bacteria [42; 43; 44]. Recent studies have identified many bacteria-derived biomolecules, including toxin proteins, as OMV cargoes, and it has been reported that OMVs and their cargoes induce various biological effects in host cells [45; 46; 47; 48].