*2.4.2 Genes and regulation*

While some *Vibrio* species (*V. parahaemolyticus*) possess two T3SS (T3SS1 and T3SS2), only one, with similarities to the T3SS2 of *V. parahaemolyticus*, has been found in *V. cholerae*'s genome [111]. The T3SS genes are located on a genomic island of approximatively 49kb, which includes an integrase, structural components,

#### *The Secretome of* Vibrio cholerae *DOI: http://dx.doi.org/10.5772/intechopen.96803*

effectors and regulators (*vttR*A, *vttR*B) [114]. The T3SS genomic island is acquired by horizontal transfer [115]. The core region contains most of the structural components and some effectors, while the upstream and downstream regions, more affected by the gene transfer, harbor a variety of effectors [109]. The *Vibrio* type three regulators VttRA and VttRB share similarities with ToxR, an important virulence regulator in *V. cholerae* [116]. The regulators VttRA and ToxR control the expression of VttRB, which, afterwards, controls the expression of the T3SS structural genes in presence of bile [117]. The deletion of either of these regulators leads to a decreased T3SS-dependant cytotoxicity. VttRA and VttRB might also regulate genes outside the T3SS island [110].

### *2.4.3 Secreted proteins*

The presence of T3SS in non O1/O139 strains leads to intestinal epithelium damages, such as alteration of the brush border and disruption, as seen in the infant rabbit model of infection [112]. It is the result of the translocation of many effectors into the eukaryotic host cytoplasm by the T3SS. In *V. cholerae*, there are 7 effectors encoded within the T3SS core genomic island and at least 5 others have been identified in the up and downstream regions [109]. The first effector to be identify is *Vibrio* outer protein F (VopF - NT01VC2350) [118, 119]. VopF possesses 2 actin binding domains, the formin homology-1 like and WASP homology 2 domains, that intervene in actin polymerization of the host intestinal epithelial cells. It has been shown to be essential for virulence in infant mouse model of infection [119]. VopF has a homolog in other non O1/O139 strains, VopN, that shares 55% similarity [119]. Just like VopF, VopN disturbs actin polymerization by nucleation, but unlike VopF, locates in the stress fibers by binding filamin. Both would also have an anti-apoptotic effect.

A total of 11 proteins that use the T3SS for their secretion have been identified by using a FRET technique to visualize the translocation of proteins in HeLa cells, including an effector specific to *V. cholerae,* VopX (A33\_1663) [114]. VopX has been found to be essential for colonization in infant mouse model of infection and to induce an important growth defect in *S. cerevisiae* by destabilization of the cell wall through Cell Wall Integrity MAP kinase pathway activation [114, 120].

Another of the secreted effectors is VopE (A33\_1662) [121]. VopE is translocated to the mitochondria after its secretion by the T3SS, where it acts as a GTPaseactivating protein. Its presence in the mitochondria intervenes with the normal process of Rho GTPases Miro1 and 2, thus with the immune response using mitochondrial signalisation pathways [121, 122]. Along with VopF, VopE would lead to the loosening of the tight junctions, a primordial structure of the intestinal epithelium [119]. VopM (A33\_1684) is another effector secreted by the T3SS that leads to actin stress fibers formation and brush border effacement [110].

Other effectors have been identified, but their function remains unclear, such as VopZ (A33\_1704), VopW (A33\_1690), VopA (A33\_1680), VopG (A33\_1697), VopI (A33\_1687), VopY (A33\_1700), VopH (A33\_1678) and VopK (A33\_1699) [110, 114]. VopW is known as a hydrophilic translocator that would both have structural and effector roles [114]. Despite the lack of information, a study on multiple effectors brought some light on their potential role in infection [110]. It stated that VopA, VopM, VopW and VopH seemed to be required for intestinal colonization in infant mouse model of infection, as mutants of these effectors where not recovered from infected animals. VopA could also have a role in adhesion to the intestinal cells in the early stages of infection. Along with VopH, VopI and VopW, VopA could be part of the structural apparatus as it is essential for other effectors secretion.

#### **2.5 Type IV secretion system, a crucial virulence factor**

Three T4Ps can be found at the surface of *V. cholerae*, TCP, the chitin regulated pilus (ChiRP) and the mannose sensitive hemagglutinin pilus (MSHA). T4P have structural similarities with the T2SS, and their structure has been reviewed elsewhere [123]. An inner membrane complex, docking an ATPase cytoplasmic complex, recruits a secretion pore in the outer membrane. The pilin subunits are then assembled and secreted to form a strong but malleable filament. They have a role in many biological processes leading to virulence such as, in *V. cholerae*, acquisition of mobile genetic elements (MGE), micro-colonies formation in the intestinal lumen, adhesion to abiotic surfaces or chitin and biofilm formation [123]. The bacterial aggregation by pilus-pilus interaction with TCP, in form of micro-colonies, allows concentration of the toxin at the site of colonization and protection of the immune system (as would a biofilm) [124]. Most T4Ps have cytoplasmic ATPases that allow their elongation and retraction, which can lead to eDNA capture and motility. The main secreted components of the T4P are the pilin subunits.

### *2.5.1 The toxin coregulated pilus*

The pandemic virulence potential of *V. cholerae* resides in its MGE, harbouring both the CT and TCP apparel on the integrated CTXφ phage and *Vibrio* Pathogenic Island 1 (VPI-1), respectively [125]. TCP is essential for effective colonization of the intestinal epithelium in pandemic O1/O139 strains [126]. The VPI-1 harbours the receptor for the CTXφ phage, the major pilin of TCP (TcpA), allowing its entry into *V. cholerae*. It also regulates the CT production with ToxT, which also regulates TCP expression [48, 125]. It is believed that acquisition of both these MGE is enough to convert environmental strains into pathogenic strains [125]. Considering this information, it is clear that the gene acquisition by horizontal transfer is important for the toxigenic potential of *V. cholerae*. Obviously, TcpA, being the major component of the filament, is responsible for the pilus:pilus interaction that leads to the formation of the micro-colonies [124]. TCP also has a minor pilin, TcpB, which is also secreted and initiates pilus polymerization and retraction, despite the lack of a retraction ATPase in TCP [127]. TcpB would also bind to CTXφ minor coat protein and then leads to its internalization into *V. cholerae* by initializing the retraction of the T4P [127].

#### *2.5.2 The mannose sensitive hemagglutinin pilus*

MSHA is produced by O1 El Tor and O139 strains, but not by the O1 classical strains, and is important for adhesion to chitinous surface and biofilm formation, although it does not seem to play a role in virulence nor colonization in humans [27, 31, 126, 128]. Its filament is composed of the single major pilin MshA [129]. The dynamic of retraction/polymerization of the MSHA is controlled by c-di-GMP [129].

#### *2.5.3 The chitin regulated pilus*

The third T4P identified in *V. cholerae* is ChiRP [31]. Because, in its marine life, *V. cholerae* can use chitin as a carbon source, the capacity to colonize shellfish is then primordial to acquire this element. The expression of PilA, the major pilin of ChiRP, is induced when the bacteria are grown in presence of chitin [31]. The absence of PilA, thus of ChiRP, decreases, but does not suppress, the ability of *V. cholerae* to colonize crab shell, even though it has no effect in infant mouse model nor on adhesion to human cells [130]. It suggests that ChiRP has a role in adhesion to chitin in collaboration with other chitin binding structures and proteins (MSHA, GbpA). The colocalization of ChiRP at the pole of *V. cholerae* along with the T2SS, secreting chitinases required for chitin acquisition, would increase the effectiveness of chitin uptake by limiting the secretion to an adhesion site [31]. In other *Vibrio*, ChiRP could also have a role in biofilm formation by mediating bacterium:bacterium interactions, a phenomenon that has also been observed in *V. cholerae* and that could further increase chitin uptake [131, 132]. It is important to note that the chitin utilization pathway is linked with natural competence pathway and that ChiRP is implied in eDNA uptake [131]. eDNA uptake is used by bacteria to gain new functions, such as virulence and resistance factors, and to increase their fitness and survival in environment.
