**4.1 From motility to initial adhesion**

Planktonic *V. cholerae* are motile cells and their motility is ensured by a single polar flagellum. The flagellum is composed of three major structural components: i) the basal body, ii) the hook and iii) the filament (for detailed structure: [165]). Structurally, the flagellum is closely related to the T3SS and therefore has been referred as fT3SS (flagellum Type-3 Secretion System) [166]. The main proteins secreted through the fT3SS are the flagellins. In *V. cholerae*, five different flagellins (FlaA-E) encoded on two chromosomally distinct loci (*flaAC and flaEDB)* have been described. From these 5 flagellins, only FlaA is essential for the motility [167]. Recently, an elegant study from Dongre *and coll*. demonstrated that the cytotoxin MakA is also secreted through the fT3SS [168]. This toxin is involved in colonization and virulence in zebrafish and *C. elegans* infection models and represents the first characterization of a toxin secreted through the fT3SS in *V. cholerae* [168].

*V. cholerae* motility and chemotaxis are important for the virulence in the human gut [169]. They are necessary for the bacteria to travel from the lumen to the epithelial cells, where the virulence factors are secreted after attachment of the bacteria. Motility also plays a key role in biofilm formation on surfaces as an essential element of the initial adhesion of the bacteria. On abiotic surfaces, *V. cholerae*'s adhesion involves MSHA to "scan the surface" [170]. The flagellum is responsible for the

bacterial rotation on the surface of the support, which allows MSHA to reach a spot of high affinity. This adhesion is signaled through the flagellum rotor and results in an inhibition of motility pathways and the secretion of the *Vibrio* polysaccharides (VPS) [171]. Two other T4Ps are implicated in bacterial adhesion; ChiRP and TCP (see T4SS section). Initial adhesion to biotic surface is promoted by GbpA, which is secreted through the T2SS and induces mucin secretion [21].

Additionally, FrhA (hemagglutinin) and CraA (adhesin) secreted through the T1SS are involved in adhesion and biofilm formation (see T1SS section). The expression of both *frhA* and *craA* is controlled by a c-di-GMP-dependent regulatory system [108]. In addition, *frhA* is regulated by the flagella regulation pathways, reinforcing the role of flagella in the initial stages of biofilm formation [106].

#### **4.2 Biofilm maturation**

Once attached, *Vibrio* starts secreting VPS, which represent more than 50% of matrix composition. VPS polymers are secreted throughout the biofilm production and are mostly composed of [→4)-α-L-Gul*p*NAcAGly3OAc-(1→4)-β-D-Glc*p*- (1→4)-α-d-Glc*p*-(1→4)-α-D-Gal*p*-(1→] subunits. A variant representing around 20% of VPS consists on the same pattern except for the α-D-Glc*p* moiety that is replaced by α-D-Glc*p*NAc [172]. The VPS biogenesis and export systems are encoded on two clusters: VpsI and VpsII, encoding VpsA-K and VpsL-Q, respectively [173, 174]. Among the 18 *vps* genes, 15 of them induced highly impaired biofilm formation in *V. cholerae* when suppressed [174]. Recently, a model has been proposed for the production and secretion of VPS. In this model, the VPS are synthesized by formation and polymerization of individual subunits. The polymers are then transferred across the outer membrane through VpsM/N [175]. This system is tightly controlled by the tyrosine phosphoregulatory system VpsO/VpsU, which controls the level of phosphorylation of the C-terminal tyrosine-cluster of VpsO. High level of phosphorylation results in VpsO oligomer dissociation and VPS production reduction, whereas low level of VpsO phosphorylation results in high oligomerization and increased VPS production [175].

Shortly after VPS secretion has been initiated, the sequential secretion of the 3 major biofilm matrix proteins through the T2SS occurs. The first matrix protein to be secreted is RbmA, followed by Bap1 and RbmC [176]. More specifically, RmbC has a role in maintaining and stabilizing the biofilm [177]. A study using mutants lacking RbmC and its homolog Bap1 showed a change of colonial morphology and the loss of biofilm formation capacity [177, 178]. On the other hand, RbmA controls the structure of the biofilm [9, 179].

Growth of the biofilm is ensured by two different processes: (i) the bacteria inside the matrix are dividing inside an envelope formed by the VPS, RbmC and Bap1 [176] and (ii) new bacteria are recruited inside the biofilm. This recruitment requires the cleavage of the N-terminal domain of RbmA by PrtV. Once cleaved, RbmA can bind bacterial cells that are not producing VPS (planktonic) and recruits them into the biofilm [180]. Since MV have been observed in the biofilm matrix and PrtV can be associated to the surface of the MV, it is possible that MV play an important role in biofilm maturation in *V. cholerae* [181]. In addition, the association of Bap1 to the MV in specific conditions is likely to lead to the adhesion of the MV to the surface and to the exopolysaccharides [181]. Three other proteins with no role in biofilm formation and adhesion have been identified in biofilm preparation, *i.e*. the hemolysin HylA, HA/P and ChiA-2 [182].

Besides VPS, proteins and MV, a significant amount of eDNA is entrapped in the biofilm matrix. The roles of eDNA in bacterial physiology have been reviewed

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

elsewhere and include nutrient source, horizontal gene transfer and adherence [183]. Recently, a role in the tridimensional matrix structure of the biofilm in *V. cholerae* has been reported [184]. The eDNA inside the biofilm most probably results from cell autolysis and MV secretion [185].

## **4.3 Biofilm dispersion and detachment**

Biofilm dispersal is a complex process by which bacteria actively succeed to evade biofilm matrix [186]. Conversely to adhesion and biofilm maturation, little is known about the dispersion process of *V. cholerae* biofilms. It requires specific environmental signals, which trigger the quorum sensing and the general stress response pathways [187], matrix degradation and motility resumption [188]. The matrix degradation requires RbmB, an extracellular polysaccharide lyase that digests the VPS, and LapG, a periplasmic protease that cleaves the adhesins FrhA and CraA located at the outer membrane [188]. Under substrate specific conditions, the extracellular protease HA/P also participates in biofilm dispersion by degrading the mucin [189]. Finally, secreted nucleases such as Dns and Xds have a role in biofilm dispersion by cleaving the eDNA present in the matrix [185]. The motility resumption requires the ability to switch the flagella rotation from counterclockwise to clockwise direction mediated by CheY3 independently of chemotaxis [188].

#### **5. Conclusion**

Over the last decades, numerous studies have focused on the secreted molecules and secretion systems used by *V. cholerae* to deliver extracellular effectors. Various roles have been assigned to the secreted molecules especially regarding the host colonization and virulence, and the environmental survival and persistence, denoting their importance in *V. cholerae*'s pathogenesis and life cycle. Additionally, the redundancy of some functions carried by multiple secreted effectors testifies of their importance. With gene acquisition, MGE, strain sequencing and the emergence of more efficient technologies, it is most likely that additional secreted effectors and secretion systems will be identified and characterized.

The recent characterization of the MakA toxin secretion through the fT3SS [168] and the numerous studies on the T6SS since its discovery 15 years ago [68] clearly demonstrate that there is still work to do on the secretome and secretion systems in *V. cholerae*. The secretion mechanism of some extracellular proteins - with characterized functions - remains to be determined. It is the case of ChxA, Ace, RbmB and the DNAse Dns.

The regulation of the secretion systems and their cargo molecules is a complex process. It involves numerous regulators that can be activated or repressed depending on the detection of specific intracellular and extracellular signals. So far, most of the studies aiming to decipher the regulation pathways have been performed under laboratory conditions. The featuring of conditions that characterize the intestinal environment before and during diarrhea, including the peristaltic movement, anaerobia, the presence of the microbiota, water efflux and high osmolarity, is likely to modify *V. cholerae* secretion in terms of regulation and nature, abundance and activity of the secreted molecules. Therefore, it would be highly beneficial to study the secretion mechanisms, the secreted molecules and their regulation in models that are closer to the physiological conditions encountered in the host, such as *ex vivo* devices. Understanding the regulation and the mechanisms of colonization, virulence and resistance in these physiological conditions is crucial for the development of treatments and vaccines against *V. cholerae*.
