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.
- Vibrio cholerae
- secretion system
- membrane vesicles
2. Secretion systems
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
The growth defect of mutants lacking essential components or regulators of the T2SS shows that it is a vital component for
2.1.1 Structure and secretion through the T2SS
The structural components  and secreted proteins  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 . 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 .
2.1.2 Genes and regulation
The T2SS apparatus is composed of a dozen types of proteins, which are encoded on the
2.1.3 Secreted proteins
In the aquatic environment, after binding to zooplankton, copepods and insect egg masses,
Besides chitin, collagen can also be used as carbon source by
After the ingestion,
A second important component of the mucinase complex is the neuramidase (VCNA - VC1784). The sialidase, or neuraminidase, is encoded on the pathogenicity island of every toxigenic
The CT (VC1456-57) is an AB5 toxin secreted by the T2SS in the intestinal lumen, which represents the main virulence factor of
Prior to GM1 binding, the CT must be processed by extracellular proteases to be activated. These proteases are therefore important for virulence and colonization. Besides their capacity to activate the toxin, they have a role in finding a substrate (modification of integrin) and nutrients, and in deactivating host defense mechanisms. Among the T2SS secreted proteins, 3 serine proteases with 30% homology between them have been identified, the
Other virulence factors secreted by the T2SS have been identified in
The cytolytic toxin cytolysin/hemolysin A (VCC or HlyA - VCA0219), is secreted by the T2SS . All
Leucine aminopeptidase (Lap - VCA0812) and aminopeptidase (LapX - VCA0813) are other secreted proteases using T2SS . Lap is a zinc dependant metallo-exopeptidase that cleaves leucin in N-terminal position, while the role of LapX remains unknown . Both Lap and LapX have no role in virulence in a
Finally, several proteins involved in biofilm formation and dissemination are also secreted by the T2SS in
2.2 Type VI secretion system for competition and DNA acquisition
The type VI secretion system (T6SS) is a versatile syringe-like apparatus with homology to the phage T4 and produced by more than 25% Gram-negative bacteria that, upon contact with a target cell, punctures its cell wall, allowing translocation of toxic effectors directly into the neighboring cells [64, 65]. The cellular targets of these effectors are multiple; peptidoglycan, actin, cellular membrane, nucleic acids and immune system components, for instance . As the target cells release their DNA into the extracellular milieu upon lysis, another function of the T6SS is to capture the extracellular DNA (eDNA) in order to acquire new features such as antibiotic resistance factors and new effectors or immunity proteins . Bacteria use this device as a competition effector to take over the environmental niche and a single bacterium can possess as much as 6 different types of T6SS . In
2.2.1 Structure and secretion through the T6SS
The T6SS is anchored in the cell membrane and contains 4 distinct domains; (i) the membrane complex, (ii) the baseplate, (iii) the contractile sheath and (iv) the syringe. The current knowledge on the structure of the T6SS have been reviewed elsewhere .
Valine glycine rich proteins G 1, 2 and 3 (VgrG1-3) and a single proline-alanine-alanine-arginine repeated motif protein (PAAR) form the tip of the syringe . There are multiple PAAR proteins in
2.2.2 Genes and regulation
The complexity of the apparatus and its organization require a fine regulation to insure its efficiency and recycling. The transcriptional regulation of the T6SS in
2.2.3 Secreted proteins
As mentioned before, the T6SS apparatus carries toxic effectors directly into the target bacterial or eukaryotic cells. A single contraction event allows the translocation of many of these effectors at the same time into the target cell . The cellular targets for these effectors are multiple; they go from peptidoglycan to cellular membrane, actin and nucleic acids . To protect themselves against the toxic effectors they produce, bacterial cells express immunity proteins, which brings the notion of strains compatibility (for more information see: ). The secreted effectors and structure components can be reused by recipient cells to form a new T6SS .
Hcp is one of the proteins transported by the T6SS into the target cell, in addition to be part of its structure by forming the inner tube and serving as a chaperone to the effector molecules . Hcp is encoded by two different yet functional genes (VC1415; VCA0017) producing the same protein [68, 84]. Both genes must be knocked out to suppress the T6SS activity . Hcp is co-expressed with HlyA, and its secretion was observed before the discovery of the T6SS .
Similarly to Hcp, the VgrG proteins (VC1416; VCA0018; VCA0123) are part of the T6SS structure and are secreted into the target cell upon contraction of the T6SS [68, 85]. VgrG-1 has an actin cross-linking activity in eukaryotic cells, thus preventing cytoskeleton reorganization and phagocytosis . VgrG-2 is homologous to VgrG-1, but without a functional C-terminal effector domain . Both appear to be essential for secretion of other T6SS components as a mutational inactivation of one of these gene makes the mutant unable to secrete any T6SS-dependant effectors . Since its toxicity is exclusive to eukaryotic cells, no immunity coupled protein is required against VgrG-1. The VgrG-3 protein is known to be active against other bacteria by hydrolyzing peptidoglycan with its lysozyme-like domain, after a translocation to the periplasm [85, 87]. It might also have a muramidase activity, which could be useful in its aquatic niche to gain access to chitin or in infection to cross mucin . TsiV3 (VCA0124) acts as the antitoxin for VgrG-3 by biding to it and prevents the degradation of the cell wall in the predator bacteria . Thus, VgrG-3 might be important for infection by killing gut microbiota and by hydrolysing mucin.
The PAAR proteins (VCA0284; VCA0105), along with VgrGs, form the tip of the syringe of T6SS, bind the effectors and are therefore essential for T6SS effectors’ secretion. There are two proteins with a PAAR domain in
The cargo effector VasX (VCA0020) acts as a colicin and targets the inner bacterial membrane or eukaryotic membrane in which it is believed to form pores, increase permeability and lead to its disruption . It is encoded downstream of Hcp-2 and VgrG-2 and is regulated by VasH . Its immunity coupled protein is TsiV2 (VCA0021) . The VasW (VCA0019) protein encoded right upstream VasX is an adaptor protein that plays a role in secretion of VasX and an accessory role to VasX bactericidal activity .
The type six effector Lipase (TseL - VC1418) is another cargo effector and its secretion depends on the presence of VgrG-3. It carries a phospholipase domain that is believed to cause damage to cell membranes in both eukaryotic and prokaryotic cells [88, 91]. Its immunity coupled protein is TsiV1 (VC1419).
The type six effector Hydrolase (TseH - VCA0285) is encoded next to the PAAR protein and its secretion is dependant of the T6SS . It has been shown that TseH is able to degrade peptidoglycan, a main component of the bacterial cell wall, by hydrolysis and would therefore make it an important effector as for interbacterial competition. Its immunity coupled protein is the type six immunity hydrolase (TsiH - VCA0286), which prevents cell wall degradation.
Recently, another lipase, the Type VI lipase effector
It is most likely that, as genome analysis of more
2.3 Type I secretion system, a tool for auxiliary toxins secretion
The type I secretion system (T1SS) is used by Gram-negative bacteria to secrete, in a one-step process using ATP, proteins directly into the extracellular milieu.
2.3.1 Structure and secretion through the T1SS
The most studied T1SS is the hemolysin A associated T1SS (HlyA-T1SS) from
The secreted proteins carry a C-terminal secretion signal sensed by the inner membrane proteins upon binding . The porin TolC is then recruited to the complex, and the proteins pass through the HlyB and HlyD channel. The binding of TolC to the inner membrane complex allows its opening and the secretion of the protein to the extracellular milieu, whereafter TolC leaves the complex . As the inner membrane proteins bind to specific substrates, the TolC can be used by multiple T1SS within a cell . The secreted proteins have a functional domain in N-terminal and are secreted shortly after their translation in their unfolded state. In
2.3.2 Genes and regulation
2.3.3 Secreted proteins
The repeat in toxin (RTX) proteins are a class of proteins exclusively secreted by the T1SS . They include the HlyA of
One T1SS has been described in
Three other T1SS could be found in
2.4 Type III secretion system for colonization and injection of effectors into eukaryotic host cells
The Type III secretion system (T3SS) is a multicomponent device translocating various effectors directly into the neighbouring eukaryotic host cells and is found in many
2.4.1 Structure and secretion through the T3SS
The T3SS is a multicomponent apparatus spawning both bacterial membranes. While the effectors’ secretion through T3SS is Sec independent, the translocation of the membrane components of the injectosome requires it . The T3SS uses ATP for the active translocation of the effectors through both bacterial membranes directly into host cell cytoplasm. The T3SS consists of an injectosome with structural and genetic homology to the flagellum and a molecular syringe, the structure has been reviewed elsewhere . In brief, the syringe connects the membrane complex to the host cell cytoplasm. It is composed of (i) a basal needle, (ii) a tip and, at its end, (iii) a translocation pore. The membrane complex is composed of an assembly of concentric rings creating a channel through both bacterial membranes. It includes an outer membrane ring connected, in the periplasm, to the inner membrane ring, in addition to a cytoplasmic portion, made of a cytoplasmic ring and an ATPase complex. The exact T3SS assembly in
2.4.2 Genes and regulation
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 . It is the result of the translocation of many effectors into the eukaryotic host cytoplasm by the T3SS. In
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
Another of the secreted effectors is VopE (A33_1662) . VopE is translocated to the mitochondria after its secretion by the T3SS, where it acts as a GTPase-activating 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 . VopM (A33_1684) is another effector secreted by the T3SS that leads to actin stress fibers formation and brush border effacement .
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 . Despite the lack of information, a study on multiple effectors brought some light on their potential role in infection . 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
2.5.1 The toxin coregulated pilus
The pandemic virulence potential of
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 . The dynamic of retraction/polymerization of the MSHA is controlled by c-di-GMP .
2.5.3 The chitin regulated pilus
The third T4P identified in
2.6 Other secreted molecules
Accessory cholera enterotoxin (Ace - VC1459) and zonula occludens toxin (Zot - VC1458) are accessory toxins that are both encoded near the CT genes on the CTXφ phage [136, 137]. Zot leads to the disruption of the tight junctions between intestinal epithelial cells, an important structure in the intestinal permeability [138, 139]. It is translocated and anchored in the outer membrane and has two functional domains. The N-terminal domain is important for CTXφ phage’s morphogenesis and the C-terminal domain is cleaved by proteases. Once released into the intestinal lumen, the C-terminal domain acts as a toxin [139, 140]. Thus, Zot does not employ a conventional secretion system for its release into the extracellular milieu. Regarding Ace, it leads to fluid secretion in rabbit ileal loop model by unbalancing calcium secretion and the secretion mechanism has not been determined yet .
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 . 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 .
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
Besides AMP, MV are also involved in serum resistance in
A role of the MV in resistance to bacteriophages has also been demonstrated in
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 . It has been demonstrated that MV are involved in the formation of biofilms in several Gram-negative bacteria . In
3.3 Membrane vesicles and virulence
The MV can also carry virulence factors including the CT, the major virulence factor of
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 , PrtV metalloprotease  and the VCC . Therefore, the MV of
4. Biofilms and flagella
Most of the bacteria, including pathogens, form biofilm to survive and persist in different environments. Biofilms are organized bacterial communities attached to a surface and producing a matrix.
4.1 From motility to initial adhesion
Additionally, FrhA (hemagglutinin) and CraA (adhesin) secreted through the T1SS are involved in adhesion and biofilm formation (see T1SS section). The expression of both
4.2 Biofilm maturation
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 . More specifically, RmbC has a role in maintaining and stabilizing the biofilm . 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  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 . 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
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 elsewhere and include nutrient source, horizontal gene transfer and adherence . Recently, a role in the tridimensional matrix structure of the biofilm in
4.3 Biofilm dispersion and detachment
Biofilm dispersal is a complex process by which bacteria actively succeed to evade biofilm matrix . Conversely to adhesion and biofilm maturation, little is known about the dispersion process of
Over the last decades, numerous studies have focused on the secreted molecules and secretion systems used by
The recent characterization of the MakA toxin secretion through the fT3SS  and the numerous studies on the T6SS since its discovery 15 years ago  clearly demonstrate that there is still work to do on the secretome and secretion systems in
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
This work was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC http://www.nserc-crsng.gc.ca/index_eng.asp) Discovery grant number RGPIN-2017-05322. AMD received financial support from the Fonds de recherche du Québec en Santé, doctoral training scholarship #290352.