**Abstract**

*Vibrio cholerae* is a facultative human pathogen responsible for the cholera disease which infects millions of people worldwide each year. *V. cholerae* is a natural inhabitant of aquatic environments and the infection usually occurs after ingestion of contaminated water or food. The virulence factors of *V. cholerae* have been extensively studied in the last decades and include the cholera toxin and the coregulated pilus. Most of the virulence factors of *V. cholerae* belong to the secretome, which corresponds to all the molecules secreted in the extracellular environment such as proteins, exopolysaccharides, extracellular DNA or membrane vesicles. In this chapter, we review the current knowledge of the secretome of *V. cholerae* and its role in virulence, colonization and resistance. In the first section, we focus on the proteins secreted through conventional secretion systems. The second and third sections emphasize on the membrane vesicles and on the secretome associated with biofilms.

**Keywords:** *Vibrio cholerae*, secretome, secretion system, membrane vesicles, biofilm

## **1. Introduction**

*Vibrio cholerae* is a Gram-negative bacterium responsible for the cholera disease, which infects millions of people per year worldwide. In the environment*, V. cholerae* is a common inhabitant of aquatic ecosystems. Over more than 200 serotypes of *V. cholerae* have been described, but only two are responsible of the pandemics, *i.e.* O1 and O139 serotypes. The O1 serotype is divided in two biotypes, classical and El Tor. The *V. cholerae* O1 El Tor is responsible for the ongoing 7th pandemic [1]. The infection usually begins with the ingestion of contaminated water or food. Once inside the human host, *V. cholerae* colonizes the small intestine where biofilm-like structures have been observed [2]. The colonization and virulence inside the host are highly correlated with the secretion of a panel of proteins, including the cholera toxin (CT). The toxin is responsible for the misfunction of the calcium channel of the host epithelial cells, leading to the cholera characteristic massive loss of water and the diarrhea [3]. This review aims to focus on the secretome of *V. cholerae* and the secretion systems used by this bacterium to colonize the human host, compete with other bacteria, and survive in the environment.

## **2. Secretion systems**

*V. cholerae* possesses as many as five multicomponent secretion systems, allowing secretion or translocation of a broad range of molecules into the extracellular milieu

or directly into the neighbouring cells. These molecules are essential for niche competition in the environment and for persistence in the host.

#### **2.1 Type II secretion system for virulence and environmental fitness**

The type II secretion system (T2SS) shares many structural characteristics with the type IV pilus (T4P) and is conserved among Gram-negative bacteria for delivery of colonization and virulence factors in the extracellular milieu [4, 5]. In *V. cholerae*, it is used in the aquatic environment and in the human host to secrete exoproteins from the periplasm to the extracellular milieu or to anchor the bacteria to the host cells [4, 6]. The loss of T2SS altered growth, biofilm formation, antimicrobial resistance, and cell envelope integrity, suggesting that the T2SS has an essential role in this bacterium, which makes it a suitable target for therapeutic development [7–9]. The T2SS genes are referred to as extracellular protein secretion (*eps*) [7]. Hydrolyzation of ATP is required to provide energy for secretion [5]. The T2SS is anchored in both bacterial membranes and is distributed all over the bacterial surface [10, 11].

The growth defect of mutants lacking essential components or regulators of the T2SS shows that it is a vital component for *V. cholerae*, mostly since all the proteins secreted by the T2SS seem to act together to facilitate *V. cholerae* colonization and survival in ecological niches or in the human host. Majority of experiments occur in controlled laboratory conditions which do not represent the complexity of the intestinal nor the marine niches. These conditions might influence the type of proteins that are secreted by the bacteria, as seen in the Sikora et al. study where the CT has not been detected in the supernatant while it is a known T2SS secreted protein [6].

#### *2.1.1 Structure and secretion through the T2SS*

The structural components [5] and secreted proteins [4] of the T2SS have recently been the object for reviews. Briefly, the T2SS assembles in 4 complexes; (i) the secretin, a pore located in the outer membrane, (ii) the inner membrane anchoring platform, (iii) the intracytoplasmic ATPase complex and (iv) the pseudopilus. Even though the exact sequence of biogenesis is still unknown, a general pathway of assembly has been suggested.

The targeted proteins with signal peptides are firstly translocated to the periplasm by Sec or Tat, where they are assembled to acquire a secretion competent conformation [12, 13]. Then, it has been proposed that they bind to the pseudopilin trimeric tip and to the inner membrane platform. This interaction activates the ATPase hydrolysis activity, thus the pseudopilus elongation by addition of pseudopilin subunits and leads to the thrust of the secreted protein through the secretin channel as a piston [5]. It has been proposed that the signals for T2SS transportation are dependent on the protein conformation on the N-terminal signal peptides, but they have not been clearly identified yet [14].

#### *2.1.2 Genes and regulation*

The T2SS apparatus is composed of a dozen types of proteins, which are encoded on the *eps* operon (*epsC* to *epsN*), plus *epsAB* and the *vcpD* (*pilD*) genes in *V. cholerae* [7, 10]. Few studies have concentrated on the regulation of the T2SS in *V. cholerae*. Under laboratory conditions, the T2SS is constitutively expressed in *V. cholerae* following the growth rate of the bacteria with a higher expression at 25°C than at 37°C [15]. In addition, studies on the T2SS regulation suggest that several major regulatory pathways, including the quorum sensing, the c-di-GMP, the σE envelope stress response, might be involved [15, 16]. Finally, as more than 20 extracellular proteins with important activities throughout the infection are secreted through the T2SS [6], this system must be tightly controlled over time to allow their synchronized secretion. Therefore, the expression of the cargo proteins is regulated by a panoply of regulators.
