**3. Membrane vesicles, the type 0 secretion system**

Most bacteria, including Gram-negative and Gram-positive bacteria, release MV, also known as the type 0 secretion system [141]. Different types of MV can be produced including the outer membrane vesicles (OMV), the outer-inner membrane vesicles (OIMV), the cytoplasmic membrane vesicles (CMV) and the tube-shaped membranous structures (TSMS). The different types of MV differ in their composition and their biogenesis mechanisms, which will not be presented here since they have already been reviewed elsewhere [142]. *V. cholerae* can secrete OMV and OIMV, which contain lipopolysaccharides, phospholipids, proteins [143], DNA and RNA [144, 145]. An hypervesiculation has been reported in *V. cholerae* at the early stages of intestine colonization by silencing the phospholipid transporter VacJ/Yrb involved in the maintenance of the outer membrane lipid asymmetry. This hypervesiculation is characterized by a drastic modification of the membrane composition and a better colonization of the host intestine [146].

*In vitro*, the protein cargo of MV is highly dependent on the bacterial growth conditions [147]. The protein cargo of the MV secreted by *V. cholerae* El Tor O1 has been determined under virulence activating conditions. A total of 90 proteins associated to MV have been identified, 50 % being outer membrane or periplasmic proteins [143]. Among these proteins, some are secreted in association with the vesicles, such as the membrane and periplasmic proteins, while others are secreted independently and associated with the vesicles in the extracellular compartment, such as Bap1 [148]. The proteins associated with the vesicles can have a role in resistance (antimicrobials, plasma and bacteriophages), in biofilm formation or in virulence.

#### **3.1 Membrane vesicles and resistance**

A role for the MV in antimicrobial peptides (AMP) resistance has been reported in several Gram-negative bacteria including *V. cholerae*. Our previous studies demonstrated that PrtV-associated MV can protect *V. cholerae* from the lysis by LL-37, a cathelicidin secreted by the epithelial cells in response to *V. cholerae* infection [56]. In addition, the matrix protein Bap1 can bind to OmpT, a porin located in the outer membrane, on the surface of the MV of *V. cholerae* in presence of polymyxin B and confer cross-resistance to LL-37 [148]. Interestingly, the hypervesiculation observed during the early stages of infection leads to a decrease of OmpT in the outer membrane correlated with an increase in OmpT abundance in the MV [146]. The authors suggest that the hypervesiculation is a process used by *V. cholerae* to quickly modify the outer membrane protein content in order to increase the intestinal colonization fitness. Therefore, the hypervesiculation *in vivo* may contribute to the Bap1 mediated AMP resistance in the intestine where analogues of polymyxin B are secreted by the microbiota. In *V. cholerae*, the expression of *ompT* is negatively correlated with the expression of *ompU* through the ToxR switch [149, 150]. During the hypervesiculation process, *ompU* expression increases, leading to an accumulation of OmpU in the membrane [148]. A role of OmpU in AMP and bile resistance has been reported in *V. cholerae* [151]. Therefore, the hypervesiculation in *V. cholerae* might represent a double advantage in terms of AMP resistance through vesicles associated OmpT-Bap1 and membrane bounded OmpU.

Besides AMP, MV are also involved in serum resistance in *V. cholerae*. Septicemia caused by *V. cholerae* has been reported, especially in patients suffering liver disorders, which can lead to a 50% mortality rates [152]. In an elegant study, Aung *et al*. demonstrated that IgG present in the serum of healthy people can recognize OmpU of *V. cholerae*, which leads to the recruitment of the complement through C1q binding and to the clearance of *V. cholerae* [153]. However, the presence of OmpU in the MV can sequester the anti-OmpU IgG before they reach the bacterial cells, leading to an increased resistance of the bacteria to serum [153].

A role of the MV in resistance to bacteriophages has also been demonstrated in *V. cholerae* [154]. The authors proposed that, similarly to Bap1 and AMP and to OmpU and IgG, the presence of MV is used as bacterial decoy to lure the phages before they can reach the bacterial cell. In this case, it is the presence of phage receptors on the surface of the MV that is responsible for the sequestration of the phages [154].

#### **3.2 Membrane vesicles and biofilm**

A significant part of antimicrobial resistance is associated with the biofilm lifestyle of bacteria. The bacteria growing in a biofilm are up to 1000 times more resistant to antimicrobials and disinfectants than their planktonic counterparts [155]. It has been demonstrated that MV are involved in the formation of biofilms in several Gram-negative bacteria [156]. In *V. cholerae*, the association of Bap1, PrtV and eDNA with the MV might have a role in biofilm formation by strengthening the structure and by recruiting planktonic bacteria. More information on the role of MV in biofilm is provided in the Biofilms and Flagella section.

#### **3.3 Membrane vesicles and virulence**

The MV can also carry virulence factors including the CT, the major virulence factor of *V. cholerae* [157]. After secretion, The B subunits interact with the GM1 receptor at the surface of the epithelial cells and the toxin is endocytosed. The A

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

subunit dissociates from the rest of the toxin in the endoplasmic reticulum and spontaneously unfold. The unfolded form of the A subunit is responsible for the toxic activity of the CT. The vesicle-associated toxin is biologically active although only A subunits are encapsulated [158]. It has been demonstrated that the MV can enter inside the host cells using different mechanisms involving clathrin coated pits, formation of caveolae, use of lipid rafts and direct fusion with host cell membrane [159]. Therefore, it is likely that the lack of B subunits is not an issue for the delivery of active A subunits of the CT while encapsulated inside the MV.

Besides the CT, other biologically active virulence factors can also be transported to the host cells through MV. It is the case for HA/P and VesC [160], PrtV metalloprotease [56] and the VCC [161]. Therefore, the MV of *V. cholerae* can carry a concentrated arsenal of virulence factors that can be efficiently delivered to the host cells and have a role in *V. cholerae* pathogenesis.
