*2.3.3 Secreted proteins*

The repeat in toxin (RTX) proteins are a class of proteins exclusively secreted by the T1SS [99]. They include the HlyA of *E. coli* (but not of *V. cholerae* – see T2SS) and RtxA and FrhA of *V. cholerae*. Briefly, these proteins contain glycine and aspartaterich sequence in C-terminal, before their T1SS secretion signal, and a functional domain originally associated with toxin activities. They require activation by the acetyl transferase activator encoded within the operon. The RTX region offers many Ca2+ binding sites. Once bound to the sites, the Ca2+ generates a sudden conformation change and formation of the secondary structure of the RTX protein. As the Ca2+ concentrations are low inside the cells, the RTX proteins keep their unfolded state until they reach the extracellular milieu, where the Ca2+ concentrations are higher.

One T1SS has been described in *V. cholerae.* It is associated to the RTX toxin (RtxA), a large toxin found in many *V. cholerae* strains, including O1 El tor, O139 and non-O1/O139 strains, but not in the O1 classical strains that contain a deletion into the gene cluster [98, 100]. The omnipresence of RtxA toxin among currently circulating strains lets us think that it is an important virulence factor that could be responsible for the non-O1/O139 strains' emergence [100]. RtxA leads to the depolarization of actin, by cross-linking the actin monomers into dimers, trimers or multimers, which causes rapid rounding of host cells [101–104].

Three other T1SS could be found in *V. cholerae*'s genome. The first one is associated to two putative RTX toxins with hemolytic activity (RtxL1 and RtxL2) that have been discovered in the genome of many *V. cholerae* strains [105]. They induce cell rounding and cytotoxicity and, unlike RtxA, also have a hemolytic activity. The locus has been identified but the secretion pathway has not, although all RTX proteins are secreted through a T1SS, the RtxL associated T1SS is yet to be described. Another of the putative T1SS of *V. cholerae* is associated to another RTX-like toxin, the Flagellum-regulated hemagglutinin A (FrhA - VC1620) [106]. The decreased hemagglutination in non-motile *V. cholerae* mutant has led to the discovery of FrhA. FrhA contains a RTX-like domain and a T1SS signal and has a role in hemagglutination, adhesion to human host cells and chitin, thus in colonization and biofilm formation [107]. Its regulation is comprised in the four-step hierarchy regulation of motility, which includes the regulation of several virulence factors. FrhA is encoded in a gene cluster harbouring components with homology to TolC (FrhC - VC1621) and HlyB (VC1628). However, no HlyD homolog has been found in the surrounding genes and the homology of VC1618 to HlyB is poor, as the ATP binding site, essential to the translocation process through the T1SS, is missing [106]. This secretion system is yet to be described. The retention module-containing protein (CraA - VCA0849) also contains a glycine rich module used as T1SS secretion signal, but its secretion system remains to be described [108]. CraA is an adhesin that has a role in early stage of biofilm formation by binding chitin. It has some homology to RtxA. In other Gram-negative bacteria, it serves as an adhesin on the bacterial surface.
