Mucosal surfaces such as skin and gill are primary adhesion sites for several Flavobacteriaceae such a F. columnare, F. branchiophilum, F. spartansii, and T. maritimum. Several adhesin candidates have been identified, including surface lipopolysaccharides, capsules and fimbriae (Magarinos et al., 1995; Ostland et al., 1997; Klesius et al., 2008; Loch and Faisal, 2014; Pérez-Pascual et al., 2017). In addition, adherence based on hemagglutinins and lectin-like carbohydrates that are part of the capsule are responsible for attachment to gill tissue in F. columnare (Decostere et al., 1999; Bernardet and Bowman, 2006). Flavobacterium psychrophilum seem to adhere to gill tissue using several leucine-rich repeat proteins, similar to BspA and LrrA of bacteroidetes causing periodontal disease (Sharma et al., 1998; Kondo et al., 2002; Nematollahi et al., 2003a). Flavobacterium psychrophilum adheres to stomach and intestinal mucosa in rainbow trout (Nikoskelainen et al., 2001). In both F. psychrophilum and F. columnare the ability to agglutinate, adhere to, and hemolyze rainbow trout erythrocytes is associated with a sialic acid-binding lectin found in some serotypes of the pathogen (Lorenzen et al., 1997; Møller et al., 2003). The adherence of F. branchiophilum on mucosal surfaces of gills is the initial stage of infection in gill disease and this may be facilitated by several adhesin genes horizontally acquired by the pathogen (Ostland et al., 1997; Touchon et al., 2011).

Several adhesins of members of the Flavobacteriaceae family are secreted through T9SSs (Touchon et al., 2011; Pérez-Pascual et al., 2017; Barbier et al., 2020; Kunttu et al., 2021). Flavobacterium columnare, F. psychrophilum, F. spartansii, T. maritimum, possess genes involved in gliding motility, an active process independent of pili or flagella (Perry, 1973; Alvarez et al., 2006; Touchon et al., 2011; Loch and Faisal, 2014; Pérez-Pascual et al., 2017; Penttinen et al., 2018). Flavobacterium columnare, F. spartansii, T. maritimum and F. psychrophilum contain T9SS-associated genes such as gld and spr, that are involved in gliding motility. It has been shown that deletion of gld genes such as gldD, gldG and gldN not only leads to deficits regarding the ability of gliding motility and adhesion, but also negatively affects biofilm formation and extracellular hemolytic and proteolytic activities, indicating that T9SSs are involved in both gliding motility and secretion of proteins (Sato et al., 2010; Touchon et al., 2011; McBride and Nakane, 2015; Chen et al., 2017; Li et al., 2017; Pérez-Pascual et al., 2017; Barbier et al., 2020; Kumru et al., 2020). Despite the unique gliding motility properties of numerous bacteroidetes, some member species of the family such as F. branchiophilum, T. maritimum are reported to utilize a non-gliding motility mechanism as they contain pili, fimbriae or pili-like structures on their cell surface (Heo et al., 1990; Rahman et al., 2014).

Pathogens have evolved different mechanisms to overcome bacterial competition in order to secure efficient colonization of their host. Among the bacteroidetes, that are the overall most dominant phylum of animal gut microbiota, F. johnsoniae is known to possess a unique T6SS named T6SSⁱⁱⁱ which is directly involved in bacterial competition (Russell et al., 2014b). Similarly, genomic analysis of some strains of F. branchiophilum, F. spartansii, and F. columnare show the presence of T6SSⁱⁱⁱ coding genes that might be involved in host bacterial competition (Abby et al., 2016; Kumru et al., 2017, 2020).

Furthermore, bacteroidetes evolved diverse survival mechanisms to escape the hostile environments generated by host cells to hinder their colonization. These include defense mechanisms against bacterial pathogens such as the production of reactive oxygen species (ROS) by host macrophages (Secombes, 1996). Bacteroidetes harbor genes encoding superoxide dismutase, catalase-peroxidase, and thiol peroxidase to resist ROS-mediated killing (Duchaud et al., 2007; Chen et al., 2017; Levipan et al., 2018). Iron acquisition systems have been identified in F. columnare (Guan et al., 2013; Zhang et al., 2017), F. psychrophilum (Møller et al., 2005), F. spartansii (Chen et al., 2017), F. branchiophilum, and T. maritimum (Avendaño-Herrera et al., 2005), whose function is to compete for the limited iron stock in host cells. The uptake of iron-siderophore complexes by bacteroidetes are not fully explored, however, several TonB-dependent outer membrane receptors such as, OmpA related proteins, ferrichrome-iron receptor precursor (FhuA) and ferric uptake receptor (Fur) protein are identified in the genomes of F. columnare (Dumpala et al., 2010; Guan et al., 2013), F. psychrophilum (Alvarez et al., 2008) T. maritimum (Avendaño-Herrera et al., 2005) that may function as iron importer across the outer membrane as is shown for several Gram-negative bacteria (Letain and Postle, 1997; Braun et al., 1998).

Secretion of exopolysaccharides by pathogenic bacteria provides a beneficial environment for survival in the host. F. columnare secretes large quantities of exopolysaccharides during infection using polysaccharide exporters that transport polysaccharides from the cytoplasm to the periplasm (Zhang et al., 2017).

Fish-pathogenic flavobacteriaceae isolated from different fish species produce several secreted proteases with elastase, gelatinase, collagenase, and caseinase activities that may have significant roles in virulence (Bertolini et al., 1994; Dalsgaard and Madsen, 2000; Ostland et al., 2000; Duchaud et al., 2007). The two economically important bacteroidetes F. psychrophilum and F. columnare cause severe mortality associated with hemorrhagic septicemia and columnaris, respectively, by using multi-factorial virulence factors including the metalloproteases (Griffin, 1987; Secades et al., 2001, 2003). Fpp1 and Fpp2, which are calcium dependent proteases that cleave protein-rich extracellular matrices of connective and muscular tissues during colonization (Secades et al., 2001, 2003). Fpp1 and Fpp2 contain the conserved carboxy terminal domain (CTD-domain) for protein secretion through T9SS, which suggest that the T9SS is the secretion system for these metalloproteases (Li et al., 2017; Barbier et al., 2020).

Virulence-associated collagenases are among the conserved virulence factors in F. spartansii (Chen et al., 2017), F. columnare (Olivares-Fuster and Arias, 2008), F. psychrophilum (Nakayama et al., 2016), and T, maritimum (Pérez-Pascual et al., 2017). They are used by the pathogens to disrupt collagen-rich tissues of the host. The specific secretion pathways for these collagenases are yet to be explored, however, different studies show the dependency of secreted proteolytic enzymes on T9SS (Chen et al., 2017; Li et al., 2017; Pérez-Pascual et al., 2017).

Polysaccharide lyases such as Chondroitin AC lyase that degrade complex acidic polysaccharides such as hyaluronic acid and chondroitin sulfates located mainly on the extra-cellar matrix of fish tissues are potential virulence factors in F. columnare, where this has been shown in vivo using rainbow trout (Stringer-Roth et al., 2002; Li et al., 2017) and in T. maritimum where the activity was tested on chondroitin sulfate-supplemented agar (Pérez-Pascual et al., 2017). These enzymes are suggested to initiate and promote infection and are secreted by T9SS: mutational deactivation of a component of T9SS, gldN, in F. columnare lead to the loss of the ability to degrade chondroitin (Li et al., 2017; Pérez-Pascual et al., 2017).

Extracellular elastases produced by F. psychrophilum may have roles in the digestion of host tissues (Madsen and Dalsgaard, 1998; Nematollahi et al., 2003b; Rochat et al., 2019). Unlike for most other virulence factors of fish-pathogenic flavobacteriaceae the secretion pathway of elastase from F. psychrophilum is T9SS-independent (Rochat et al., 2019). Yet, the specific secretion system for the elastase is unidentified.

Glycoproteins are described to be involved in pathogenesis in several pathogenic Gram-negative bacteria (Guerry, 2007), taking part in processes such as cell adhesion and motility (Virji, 1997; Young et al., 2002; Grass et al., 2003; Szymanski et al., 2003). In F. psychrophilum, several glycoproteins were previously identified and predicted to promote infection (Merle et al., 2003; Dumetz et al., 2007). An outer membrane glycosyltransferase, FpgA, expressed by F. psychrophilum has been shown to promote polysaccharide-mediated gliding motility and bacteria-host cell interactions (Fernández-Gómez et al., 2013; Pérez-Pascual et al., 2015). Mutations in the fpgA gene not only impair pathogenicity, but also downregulate expression of the metalloproteases Fpp2 and Fpp1 discussed earlier in this section (Pérez-Pascual et al., 2015). However, neither the secretion apparatus for the metalloproteases Fpp1 and Fpp2 nor its connection to the enzyme FpgA itself is understood.

Hemolysins are potent virulence factors for fish pathogens such as F. psychrophilum, F. columnare, F. spartansii and T. maritimum. They are involved in the lysis of host erythrocytes and in recovering their hemoglobin-bound iron pool for growth (Avendaño-Herrera et al., 2005; Møller et al., 2005; Högfors-Rönnholm and Wiklund, 2010; Chen et al., 2017; Kayansamruaj et al., 2017). Hemolytic enzymes play a part in the virulence of F. psychrophilum (Högfors-Rönnholm and Wiklund, 2010). F. psychrophilum contains a putative hemolysin gene, FP0063, with 53% similarity with the hemolysin of Vibrio anguillarum VAH5 (see the Vibrio section below). The Vibrio hemolysin has been shown to degrade erythrocytes creating a devastative host cell damage (Rodkhum et al., 2005; Duchaud et al., 2007). Similarly, F. spartansii harbors virulence-related hemolysin genes that are vital in pathogenesis process such as tissue damage and sepsis (Chen et al., 2017). Flavobacterium columnare contain hemolysin genes that are prominent for pathogenesis (Kayansamruaj et al., 2017; Kumru et al., 2017). Tenacibaculum maritimum contains a gene encoding for sphingomyelinase, a toxin directly involved in hemolysis (Oda et al., 2010; Pérez-Pascual et al., 2017). The secretion mechanisms of hemolysins of fish-pathogenic flavobacteriaceae is not yet clear. However, mutations affecting genome regions encoding T9SS responsible for gliding motility, including the examples GldJ, GldK, GldM, and GldN from F. psychrophilum led to reduced hemolytic activity, indicating that the secretion of hemolysins is at least indirectly influenced by T9SS (Castillo et al., 2015; Barbier et al., 2020).

Flavobacterium spartansii harbors genes encoding for toxin thiol-activated cytolysin (TACY) which possibly modulate or lyse the function of enterocyte membranes through formation of pore (Chen et al., 2017). Flavobacterium branchiophilum produces a cholera-like toxin protein, identified as FBFL15_0919 (Touchon et al., 2011), which has high sequence similarity with the cholera toxin (CTA) produced by V. cholera and the heat-labile toxin (LTA) produced by enterotoxigenic Escherichia coli. The LTA and CTA toxins stimulate adenylate cyclase and provoke the massive loss of fluid and electrolytes through the intestinal epithelium of the host (Sixma et al., 1991; de Haan and Hirst, 2000; Broeck et al., 2007). Similarly, FBFL15_0919 is speculated to disturb the osmoregulatory function of the epithelial cells of the gills. Gill cells are involved in excretion of urea and in uptake of salts, in addition to oxygen uptake (Wilkie, 2002). The exact secretion mechanism for FBFL15_0919 is not yet investigated, however, CTA is reported to be exported by T2SS (Robien et al., 2003).

Despite the availability of genome sequences of fish pathogens such as F. psychrophilum, many of the secretion systems for virulence factors that are key to the pathogenesis of other Proteobacteria are not yet described in detail in Flavobacteriaceae. Genes for key components for secretion systems from almost all classes have been found in the genomes of different Flavobacterium species, but have not yet been directly linked to virulence experimentally (Kumru et al., 2020). Much more is known about the components for T9SSs (Duchaud et al., 2007). Mutation of genes involved in gliding (the gld genes), which are components of the T9SS in F. johnsonia (a well-studied member of the fish-pathogenic bacteroidetes) lead not only deterioration of motility but also to significant changes in the secretion of extracellular enzymes (McBride et al., 2003; Braun et al., 2005; Braun and McBride, 2005) strongly suggesting that the T9SS may be the most crucial virulence factor of bacteroidetes. It facilitates pathogenicity through providing multiple virulence tools for successful infection ranging from colonization to host cell disruption processes (Li et al., 2017; Penttinen et al., 2018; Barbier et al., 2020).

The main routes of transmission for vibriosis include direct contact (entry through skin and/or damaged mucous layers) and oral (ingestion) transmissions (Grisez et al., 1996; Svendsen and Bøgwald, 1997; Jun and Woo, 2003; Weber et al., 2010). The initial stage of infection involves colonization of the skin mucosa and biofilm formation facilitated by exopolysaccharides (Croxatto et al., 2007; Weber et al., 2010). Adherence of the bacteria to host surfaces and survival in the host are particularly determined by the surface hydrophobicity of pathogenic bacteria (Lee and Yii, 1996; Balebona et al., 1998). For instance, V. anguillarum and V. salmonicida utilize surface hydrophobicity to survival in hosts organism (Horne and Baxendale, 1983; Espelid et al., 1987; Hjelmeland et al., 1988).

Vibrionaceae utilize specific pili or various non-pilus adhesins that facilitate adhesion to host cells and initiate infection. Adhesion can be mediated by flagella in V. anguillarum, V. alginolyticus, V. harveyi, V. splendidus, and V. vulnificus indicating that motility is key for adhesion in these organisms (Milton et al., 1996; O’Toole et al., 1996; Ormonde et al., 2000; Luo et al., 2016; Dai et al., 2020; Xu et al., 2021). In addition to flagella, the outer membrane protein TolC and NADH oxidase dihydrolipoamide dehydrogenase play pivotal roles in the adhesion of V. harveyi and V. splendidus, respectively (Dai et al., 2019; Zhu et al., 2019). Experiments performed on non-fish cell lines show other factors including the adhesin VpadF, enolase, capsular polysaccharide, T6SSs, multivalent adhesion molecule 7 (MAM7) and a mannose-sensitive hemagglutinin (MSHA) pilus bind to different receptors to facilitate cell attachment by V. parahaemolyticus (Hsieh et al., 2003; Krachler et al., 2011; Yu et al., 2012; O’Boyle et al., 2013; Jiang et al., 2014; Liu and Chen, 2015). A β-pore forming toxin, phobalysin, plays a pivotal role in host cell adherence in Photobacterium damselae (Rivas et al., 2015; von Hoven et al., 2018).

Survival mechanisms in the hostOnce inside the host, survival of Vibrionaceae is achieved through distinct virulence mechanisms such as regulation of iron acquisition, neutralization of acidic environments, combating oxidative stress, induction of host immune cell death, and immune evasion. V. vulnificus neutralizes acidic environments of the host gut and tolerates oxidative stress by converting lysine to cadaverine and CO₂ using lysine decarboxylase and manganese superoxide dismutase (SOD) respectively (Rhee et al., 2002; Kim et al., 2005; Jones and Oliver, 2009). Invasion and inter-microbial competition of V. cholerae in the gut of zebrafish is mediated by a T6SS where the syringe-like system plays a role in intensifying the host gut contractions which lead to expulsion of resident microorganisms (Logan et al., 2018).

Several Vibrionaceae possess finely regulated and high-affinity siderophore or heme-dependent iron acquisition systems to compete for iron sequestered by iron-binding proteins such as transferrins or in heme in host organisms (Thode et al., 2018; Richard et al., 2019). Siderophore-dependent iron acquisition systems synthesize and secrete siderophores into the environment to chelate ferric iron. The secretion mechanisms for siderophores are not fully understood in the Vibrionaceae but a major facilitator superfamily protein (MFS)-mediated efflux pump seems to play an important role (Li and Ma, 2017). No hemophores to chelate iron from hemoproteins have yet been identified in fish-pathogenic species from the Vibrionaceae family (Wandersman and Delepelaire, 2004).

Vibrio anguillarum, V. vulnificus, V. ordalii, V. harveyi, Photobacterium damselae, V. parahaemolyticus, V. alginolyticus, A. salmonicida, and V. splendidus utilize siderophore-mediated iron acquisition systems. The uptake of both ferric-siderophore complex and heme across the membrane is performed by ABC transporters which utilize TonB complex as energy transducers (Stork et al., 2004; Thode et al., 2018).

Depending on the serotype, V. anguillarum produce three distinct iron-uptake systems: anguibactin and its outer membrane receptor FatA (Lopez and Crosa, 2007; López et al., 2007; Naka et al., 2013), piscibactin and its outer membrane receptor FrpA (Balado et al., 2018; Valderrama et al., 2019) and vanchrobactin and its outer membrane transport FvtA (Soengas et al., 2006, 2008; Balado et al., 2008, 2009). Like Vibrio anguillarum, Photobacterium damselae and V. ordalii produce the piscibactin siderophore where the receptor in the former is FrpA and in the latter is unclear (Souto et al., 2012; Ruiz et al., 2019; Valderrama et al., 2019). Vibrio vulnificus synthesize vulnibactin and its uptake is mediated by VuuA (Webster and Litwin, 2000; Kim et al., 2006; Alice et al., 2008). Vibrio harveyi produce amphi-enterobactin and its uptake receptor FapA (Zane et al., 2014; Naka et al., 2018). Vibrio parahaemolyticus produces the siderophore vibrioferrin and its receptor PvuA (Funahashi et al., 2002; Tanabe et al., 2003). Like V. parahaemolyticus, V. alaginoluticus contains gene clusters for vibrioferrin biosynthesis (Wang Q. et al., 2007). Allivibrio salmonicida utilize bisucaberin that is recognized by BitA (Winkelmann et al., 2002; Kadi et al., 2008). Vibrio splendidus utilize a hydroxamate-based siderophore and IutA is proposed as a potential receptor (Song et al., 2018). Besides the siderophore iron acquisition system, V. vulnificus directly use heme from hemoglobin as host iron sources, where the uptake of heme is facilitated by outer membrane protein HupA (Litwin and Byrne, 1998). Vibrio anguillarum possess a heme-utilizing system where two outer membrane proteins, HuvA and HuvS, perform heme uptake (Mazoy et al., 1996; Mazoy and Lemos, 1996; Mouriño et al., 2005), and where the complex HuvBCD is required for subsequent transport of periplasmic heme into the cytosol (Mouriño et al., 2004).

Extracellular proteases are associated with the pathogenesis of several Vibrionacae. The extracellular zinc metalloproteases EmpA and PrtV of V. anguillarum are potent virulence factors with mucinase activities that are required for infecting the gastrointestinal tract of diverse fish species such as salmon, turbot and flounder (Norqvist et al., 1990; Milton et al., 1992; Denkin and Nelson, 1999, 2004; Zhao-lan et al., 2002; Mo et al., 2010; Frans et al., 2011). The secretion of EmpA and PrtV is mediated by a T2SS (Zhang et al., 2006; Rompikuntal et al., 2015). EmpA and PrtV belong to the M4 and M6 family of peptidases, respectively, and are translated in inactive form which passes through a maturation process involving proteolytic cleavages during secretion through the T2SS (Milton et al., 1992; Staroscik et al., 2005; Zhang et al., 2006; Varina et al., 2008; Mo et al., 2010; Rompikuntal et al., 2015). Besides its proteolytic activity, EmpA activates hemolysins promoting disruption of red blood cells in fish (Han et al., 2011). PrtV contains gelatinase, protease and glycosidase activity, and a prtV mutant of V. anguillarum strain show reduced infection, growth and hemolytic activity in turbot and turbot cell lines (Mo et al., 2010).

Vibrio parahaemolyticus produce multiple extracellular metalloproteases such as the tissue-degrading protease VPM, and the collagenases PrtV and VppC. In addition, serine proteases Vpp1/protease A with cytotoxic activity, VpSP37 with gelatinase activity, and PrtA with hemolytic and cytotoxic activity are produced as virulence factors (Osei-Adjei et al., 2018). Similarly, V. alginolyticus produce extracellular alkaline serine protease A and a VppC homolog where the former is an exotoxin and lethal to fish (Takeuchi et al., 1992). Vibrio vulnificus produce an extracellular metalloprotease Vvp, however, its involvement in virulence is debatable (Valiente et al., 2008). The secretion mechanisms of these metalloproteases are not yet described.

Six pore-forming hemolysins, Vah1-5 and Rtx, are responsible for disrupting red blood cells leading to hemorrhagic septicemia in V. anguillarum (Hirono et al., 1996; Rodkhum et al., 2005). Vah1-5 belong to the HylA hemolysin family (Hirono et al., 1996; Zhang and Austin, 2005). Secretion factors of Vah1-5 from V. anguillarum are not documented; however, HylA hemolysins are generally known to be secreted via T1SS (Thomas et al., 2014). Like the metalloproteases EmpA and PrtV of V. anguillarum, hemolysins in the HylA family are translated as pre-proteins where the production of mature hemolysins requires multiple processing steps during secretion (Zhang and Austin, 2005). All Vah hemolysins show hemolytic activity on fish erythrocytes, with Vah4 displaying the strongest virulence effects (Rodkhum et al., 2005). RtxA (repeats-in-toxin-A), a multifunctional extracellular protein secreted by diverse Gram-negative fish pathogens, is responsible for cytotoxic and hemolytic attacks in fish (Li L. et al., 2008). The secretion of unfolded Rtxs is performed by a T1SS where folding is initiated only after passage (Linhartova et al., 2018). The expression and secretion of V. vulnificus RtxA1 toxin is regulated by a general stress response regulator, RpoS (Guo et al., 2018).

Vibrio parahaemolyticus possess two pathogenic hemolysins, thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) (Li L. et al., 2019). Vibrio parahaemolyticus possess multiple secretion systems, a T2SS and two T3SSs (T3SS1 and T3SS2) where the T2SS and T3SS2 export the hemolysins. TDH possesses exotoxin roles and secreted through both, the T2SS and T3SS2 (Matsuda et al., 2019). Like TDH, TRH is secreted through both, the T2SS and T3SS2 (Matsuda et al., 2019). The pathogenicity role of T3SS1 of V. parahaemolyticus is described below.

Vibrio alginolyticus express the toxic hemolysins Tdh and TLH where the former has hemolytic activity both on mouse and fish and the latter shows both hemolytic and phospholipase activities in zebrafish (Cai et al., 2007; Jia et al., 2010). Vibrio harveyi produce a cytotoxic hemolysin VHH with phospholipase B activity, where its native secretion system is not clear; but it has been shown to use T2SS-mediated secretion when recombinantly expressed in E. coli (Zhong et al., 2006; Sun et al., 2007). Several extracellular products (ECPs) from P. damselae sp. damselae that have strong hemolytic, lipase, esterase-lipase and phospholipase activity but weak proteolytic activity are reported to be lethal for fish (Fouz et al., 1993; Labella et al., 2010).

T3SSs are ubiquitous in Gram-negative pathogens including fish pathogenic Vibrionaceae such as V. parahaemolyticus, V. salmonicida, V. alginolyticus, V. harveyi and Photobacterium damselae. They directly deliver both fish and non-fish virulence factors from the pathogen cytosol into host cells where the effect of the virulence factors on fish pathogenesis varies depending on the nature of the Vibrionaceae members (Hueck, 1998; Frey and Origgi, 2016; Osorio, 2018).

As mentioned earlier, V. parahaemolyticus harbor a set of two T3SSs (T3SS1 and T3SS2) where T3SS1 is related to cytotoxicity and T3SS2 is associated with both cytotoxic and enterotoxic activities in mice (Hiyoshi et al., 2010). Several effectors such as VopQ [that induces autophagy in mammalian cell lines; (Burdette et al., 2009)], VopS [modification of host Rho family GTPases in HeLa cells (Yarbrough et al., 2009)], VopR (unknown function) and VpA0450 [hydrolyzes plasma membrane-located phosphatidylinositol phosphate in human cells (Broberg et al., 2010)] are secreted through T3SS1. Evidence based on non-fish models show that VopA/VopP [inhibits mitogen-activating protein kinase pathway using acetyl transferase, (Trosky et al., 2007; Pan et al., 2021) VopL (induce stress fiber using actin nucleastion (Ham and Orth, 2012)], VopT [Induce cytotoxicity using ADP-ribosylation (Sun and Barbieri, 2003; Barbieri and Sun, 2004)], VopV [facilitate enterotoxicity by F-actin binding/bundling (Hiyoshi et al., 2011)] and VopC [Promote bacterial invasion using deamidase (Zhang et al., 2012)] are secreted through T3SS2 (Ham and Orth, 2012). Additional contributions of T3SS2 to pathogenicity is discussed above in the section on hemolysins.

Effectors of T3SSs in V. alginolyticus promote cell death of fish cells (Zhao et al., 2010). Val1686 and Val168 in V. alginolyticus, homologous to the T3SS1 effector proteins VoPQ (88 %) and VopS (91 %) of V. parahaemolyticus, cause apoptosis in fish cell lines (Osorio, 2018). V. alginolyticus harbor two T6SSs, T6SS1 and T6SS2, where T6SS1 secretes multiple effectors such as MIX IV pore- forming effector (Va16152), MIX I (Va01565), LytM, a peptidoglycan hydrolase and lysozyme-like domain (Va01435), OmpA_C (Va01555) and VopQ (Va17542) (Salomon et al., 2015). Also V. splendidus produce a virulence effector, Hop, with cytotoxic effects that is secreted by a T3SS (Zhuang et al., 2020, 2021).

Host cell adherence by Y. ruckeri is promoted through the pathogen’s variable motility that depends on the presence and absence of flagella (Davies and Frerichs, 1989). Bacterial adherence in Y. ruckeri may also involve type IV pili, a hair-like multi-subunit surface appendage which its function is not restricted to surface biding and twitching motility, but also DNA-uptake, and micro-colony or biofilm formation (Mattick, 2002; Craig et al., 2019).

Experimental evidence shows that the site of entry for virulent E. tarda and E. ictaluri are gills, gastrointestinal tract and body surface (Ling et al., 2001; Pirarat et al., 2016). Edwardsiella tarda is shown to involve actin and microtubules for entry to host cells (Ling et al., 2001; Sui et al., 2017). Motility is critical during the initial phase of colonization for several aquaculture pathogens (Ormonde et al., 2000). Two types, motile and non-motile strains, of E. tarda exist where there is apparent differences in the pathogenicity between these variants (Park et al., 2012), however, the difference in colonization mechanisms of these variants are not clear. Furthermore, secretion systems such as T3SS and T6SS play pivotal roles in adherence and cell-diffusion (Tan et al., 2005; Park et al., 2012). For instance, EvpP, a component of T6SS, is shown to control the internalization process in invasion suggesting that this secretion system plays a key role in the pathogenesis of E. tarda (Wang X. et al., 2009).

The site of infection of C. freundii are the mucosal layers of the intestine which suffer severe damage during infection (Pan et al., 2021). A whole genome analysis of C. freundii indicates the presence of multiple virulence factors that are potentially involved in colonization and in-host cell survival. C. freundii displays strong adhesion to the hepatic fish cell line L8824 and can cause distinctive lesions known as A/E lesions (Pan et al., 2021), similar to A/E lesions caused by E.coli on the intestinal mucosa. This effect requires strong attachment of the pathogen to the surface of the enterocyte and involves a T3SS which injects multiple effector proteins into the cell (Wong et al., 2011; Hartland and Leong, 2013). An adhesin named EaeA of C. freundii has previously been identified as key element for colonization in mice but its role in colonization of fish is not clear (Schauer and Falkow, 1993). EaeA itself is an autotransporter, a member of the type Vc secretion systems (Leo et al., 2015).

Survival mechanisms in the hostIt is crucial for pathogens to avoid host defense mechanisms, in order to survive, proliferate and maintain infection in the host. Key to Y. ruckeri survival, growth and pathogenicity in the host is its iron acquisition system that produces an enterobactin-like siderophore known as ruckerbactin; the gene encoding ruckerbactin is upregulated during infection (Fernández et al., 2004; Tobback et al., 2007). The secretion and uptake systems for ruckerbactin and ferric-ruckerbactin, respectively, are not yet elucidated in detail, however, the ruckerbactin receptor has a high degree of similarity with the ferrichrysobactin receptor of Dickeya dadantii (Fernández et al., 2004).

Edwardsiella tarda has evolved several mechanisms to survive immune responses. One of these mechanisms involves the neutralization of reactive oxygen species using redox enzymes such as superoxide dismutase (SodB) and catalase KatB (Han et al., 2006). Edwardsiella tarda has the ability to survive in host serum, to resist acidic milieu and to replicate in phagocytes with the help of a serum-induced, putative hydrogenase protein named Sip2 (Li and Sun, 2018). In serum, the complement system has a significant role in host defense against infection via mechanisms involving both innate and adaptive immunity (Walport, 2001; Merle et al., 2015). Activation of the complement system lead to bacterial membrane damage and subsequent lyses using membrane attack complex (MAC) (Endo et al., 2006; Sarma and Ward, 2011). Edwardsiella tarda has the ability to evade the bactericidal effect of host serum through blocking the activation of the complement using the zinc metalloprotease Sip1 (Zhou et al., 2015). Besides, serum enhances the tricarboxylic acid cycle of E. tarda which increases membrane potential and decreases the formation of MAC, leading to serum resistance (Cheng et al., 2017). Furthermore, the chaperone protein HtpG is shown to help E. tarda to cope with stress conditions during infection (Dang et al., 2011).

Like E. tarda, E. ictaluri has the ability to survive and grow within host macrophages (Miyazaki and Plumb, 1985; Baldwin and Newton, 1993; Pirarat et al., 2016). A T3SS translocates several effectors from E. tarda and E. ictaluri directly into the host cytoplasm, where they have essential roles for internal replication and virulence (Dubytska et al., 2016). The T3SS of E. tarda plays important roles in phagocyte survival, in host proliferation and virulence (Tan et al., 2005). For instance, the T3SS effector EseJ facilitates cell proliferation in host cells by inhibiting the oxidative stress produced by host macrophages (Xie et al., 2015). Like the T3SS, a T6SS has a pivotal role in survival and replication of E. tarda in host epithelial cells and phagocytes (Park et al., 2012). However, the mechanisms of the two secretion systems are antagonistic, where the T3SS promotes bacterial replication in host cells while the T6SS protects the pathogens from attack by the activated innate immune system through suppressing replication (Xie et al., 2015; Hu et al., 2019).

Iron acquisition systems such as siderophore biosynthesis and iron uptake systems are necessary for pathogenicity of E. tarda (Castro et al., 2016). The genome of E. tarda contains genes encoding for multiple factors putatively involved in iron utilization including ferric uptake regulator, ferric reductase, ferritin and TonB (Wang Q. et al., 2009). It also includes a gene cluster sharing high similarity to the pvsABCDE-psuA-pvuA operon which encodes proteins for synthesis and utilization of vibrioferrin siderophore of the two fish pathogen vibrios V. parahaemolyticus and V. alginolyticus (Tanabe et al., 2003; Wang Q. et al., 2007).

In contrary, there are no dedicated siderophore biosynthesis genes detected in the genome of E. ictaluri (Santander et al., 2012), however, the pathogen carries a ferric hydroxamate uptake (Fhu) system involved in uptake of hydroxamate-type siderophores. Experimental evidence shows that the Fhu-system is indeed involved in pathogenicity, suggesting that the system may be utilized to uptake hydroxamates secreted by other bacteria (Abdelhamed et al., 2016). In addition, the genome of E. ictaluri contains an alternative ferric uptake system (afuABC) and the general TonB energy transducing system (TonB-ExbB-ExbD) which may be involved in iron uptake (Abdelhamed et al., 2013, 2017). Furthermore, E. ictaluri utilizes a Fur-regulated heme-hemoglobin uptake system (Santander et al., 2012).

In addition to the siderophore-dependent systems, E. tarda can utilize hemin, hemoglobin and hematin as iron sources (Abbott and Janda, 2006). A heme utilization operon in its genome that encodes for three proteins identified as HutW_Et, HutX_Et and HutZ_Et is highly similar to the heme utilization operon in V. cholera (Shi et al., 2019). In V. cholera HutX_Vc functions as an electron carrier to the heme-degrading HutZ_Vc while HutW_Vc may be a reductase for HuyZ_Vc (Sekine et al., 2016). HutZ_Et has been shown to be involved in biofilm formation and motility (Shi et al., 2019). Furthermore, E. tarda produce an iron-mediated hemolysin that is released under iron-limited conditions (Janda and Abbott, 1993; Hirono et al., 1997). All in all, iron depletion in host cells is a signal for pathogens to turn on the expression of virulence genes. The ferric uptake regulator (Fur) protein senses iron depletion in, for example, E. tarda to regulate expression of key virulence factors such as T3SS and T6SS through secretion regulator protein EsrC (Chakraborty et al., 2011).

Citrobacter freundii harbor genes that could be utilized in diverse mechanisms and virulence factors to maintain infection, including a T3SS that plays a crucial role in the injection of effector proteins such as HopAJ2 family member that mediate survival against host immune response (Galán et al., 2014; Pan et al., 2021). Iron acquisition in C. freundii is facilitated by FepE, a component of the ferric enterobactin siderophore transport system (Wang et al., 2019). In addition to the siderophore-dependent iron transport system, an hemophore-mediated heme uptake apparatus is present that includes an outer membrane channel protein HasF (Létoffé et al., 2001). In addition, surface lipopolysaccharides (LPS) of some serogroups of C. freundii have been shown to play vital roles in colonization and host survival (Denkin and Nelson, 1999).

Yersinia ruckeri produce a serralysin metalloprotease named Yrp1 that is secreted via an ATP-dependent T1SS composed of three genes, yrpD-F, and a protease inhibitor inh. Yip1 seems to have pivotal roles in the development of ERM due to its ability to disrupt several extracellular matrix proteins of fish such as laminin and fibronectin, and proteins important in muscle function such as actin and myosin (Fernández et al., 2002). Furthermore, Y. ruckeri produce two additional peptidases, YrpA and YrpB encoded by the yrpAB operon. Toxicity tests indicate that at least YrpA is involved in Y. ruckeri pathogenicity, as deletion of the gene encoding YrpA drastically reduces the virulence of the bacteria (Navais et al., 2014).

Edwardsiella tarda produce multiple virulence-related proteases that are engaged in diverse processes of infection. For instance, the above mentioned zinc metalloprotease Sip1 of E. tarda is secreted to effectively prevent complement-mediated serum killing through blocking the activation of the complement system (Zhou et al., 2015). A serine protease autotransporter Tsh_ET, which is a temperature-sensitive hemagglutinin, belongs to the Type V secretion systems (T5SS) and contributes to the virulence of E. tarda. Tsh has multiple functions; mutations in the tsh gene led to hindrance of biofilm growth, reduction of resistance against serum killing, impairment of the ability to block the host immune response, and attenuation of tissue invasion and cellular infectivity (Hu et al., 2016). In addition, the periplasmic serine protease DegP_ET is presumably involved in virulence of E. tarda, similar to DegP in Salmonella enterica, Streptococcus pyogenes and Legionella (Johnson et al., 1991; Jones et al., 2001, 2002; Pedersen et al., 2001; Wilson et al., 2006; Flannagan et al., 2007; Jiao et al., 2010).

The gene cluster yhlBA of Y. ruckeri encodes for a hemolysin (YhlA) and a protein involved in its secretion/activation (YhlB). The expression of YhlA is upregulated in iron-limited conditions suggesting that the hemolysin may be directly involved in acquisition of iron from the host cell (Fernández et al., 2007; Wrobel et al., 2019). Both yhlA and yhlB genes have high sequence similarity with a type Vb secretion system with hemolysin activity in Serratia sp. (Wrobel et al., 2019).

Edwardsiella tarda produce two different hemolysins, the iron-mediated cell associated hemolysin EthA, secreted under iron limited conditions, and an extracellular pore-forming hemolysin district from EthA (Janda and Abbott, 1993; Chen et al., 1996; Hirono et al., 1997). Similar to the hemolysin YhlA of Y. ruckeri and its activation/secretion protein YhlB (described above), the hemolysin EthA from E. tarda needs an activation/secretion protein EthB encoded in an operon. EthA and EthB, are prevalent in hemolytic E. tarda strains isolated from diseased fish (Hirono et al., 1997) which may indicate that they are among the key virulence mechanisms utilized by the pathogenic E. tarda.

Chondroitinases are involved in the pathogenicity of infectious bacteria (Schaechter et al., 1993). Chondroitinase activity is known to be virulence factor in Edwardsiella spp. including E. tarda and E. ictaruli. These species produce chondroitinase that mediates cartilage degradation in the process of invasion (Waltman et al., 1986; Shotts and Cooper, 1996). The Sialidase NanA from E. tarda also promotes tissue invasion, and mutations of the gene encoding for the enzyme led to drastic attenuation of the pathogen, by limiting its ability for invasion and colonization (Jin et al., 2012).

Distinct strains of Y. ruckeri produce TcpA, a Toll/interleukin-1 (TIR) domain containing protein that inhibits Toll-like receptor signaling and promotes immune evasion. TcpA is also known to increase tissue damage in fish. The gene for TcpA is located adjacent to a T4SS gene cluster which may indicate that the secretion of TcpA is related to this T4SS (Liu et al., 2019). As discussed above, T3SS are a crucial and multipurpose system in E. tarda and E. ictaluri. The T3SS of E. tarda belongs to the Ssa-Esc family which includes T3SS encoded by Salmonella pathogenicity island 2 (SPI-2) in Salmonella enterica serover Typhimurium, the causative agent of foodborne illness worldwide (Anderson and Kendall, 2017). The core components of the T3SS are encoded by 34 genes, where all obtain different functions (Tan et al., 2005; Sadarangani et al., 2013). Among these are the three translocon proteins EseB, EseC and EseD that are essential for delivery of effectors into host cells (Tan et al., 2005).

The important initial infection sites for Aeromonas such as A. salmonicida include skin, gills and the gastrointestinal tract (Ringø et al., 2004; Bartkova et al., 2017). Tissue invasion in the gastrointestinal tract is mediated by extracellular virulence factors that are utilized to damage the tissue (Ringø et al., 2004). Adhesion, a prerequisite for successful colonization by pathogens, is executed by different factors in Aeromonas species. Outer membrane proteins such as OmpA and AHA1 function as adhesins in A. veronii and A. hydrophila (Fang et al., 2004; Namba et al., 2008). A tight adherence system (TaD) is present in the majority of A. hydrophila strains (Tekedar et al., 2019a). A. hydrophila and A. sobria use pili (Lallier and Daigneault, 1984; Ho et al., 1990), lipopolysaccharides (LPS) is used by A. hydrophila (Merino et al., 1996), and cell-associated lectins are used by A. veronii (Guzman-Murillo et al., 2000) to bind to host cells and tissues. Paracrystalline surface protein layers in A. salmonicida and A. hydrophila display significant hydrophobicity and contribute to adhesion (Chu et al., 1991; Beveridge et al., 1997).

Survival of A. salmonicida and A. hydrophila in the host is achieved by S-layers that bind to extracellular matrix components such as laminin, fibronectin and vibronectin providing resistance to serum killing and protease digestion (Beveridge et al., 1997; Noonan and Trust, 1997). Most Aeromonas sequester iron from their hosts through utilization of siderophore-dependent or siderophore-independent (heme-binding) mechanisms (Byers et al., 1991; Reith et al., 2008; Lange et al., 2020). The siderophore-dependent mechanisms rely on the synthesis of enterobactin or amonabactin and some A. salmonicida species are capable of synthesizing anguibactin-like siderophores (Barghouthi et al., 1989a,b; Byers et al., 1991; Telford and Raymond, 1998; Reith et al., 2008). Allivibrio salmonicida and A. hydrophila synthesize both ferric and heme iron acquisition systems (Ishiguro et al., 1986; Najimi et al., 2008b). In most cases, two catechol siderophores; acinetobactin and amonabactin, are produced simultaneously (Balado et al., 2015) and their uptake is facilitated by the TonB-dependent outer membrane proteins FstB and FstC that function as receptors for ferric-acinetobactin and ferric-amonabactin, respectively (Balado et al., 2017; Rey-Varela et al., 2019). Ferric-siderophore uptake is mediated by FstA in some species of A. bestiarum and A. salmonicida (Beaz-Hidalgo et al., 2008, 2013). An multifunctional amonabactin receptor of A. hydrophila is reported to transport several different siderophores across the membrane (Stintzi and Raymond, 2000). Heme uptake is performed by the membrane receptors HgpB in A. veronii and HutA in A. salmonicida and A. hydrophila, (Najimi et al., 2008a; Maltz et al., 2015).

Aeromonas utilize several secretion systems such as the sec-dependent systems T1SS, T2SS, and the sec independent T3SS and T6SS to transport virulence factors, toxins and effectors for infecting diverse fish species (Burr et al., 2002; Tekedar et al., 2019b). The T1SS and T2SS are prominent in almost all Aeromonas species, and a T3SS is detected in species such as A. salmonicida, A. hydrophila, and A. veronii (Citterio and Biavasco, 2015; Tekedar et al., 2019b; Barger et al., 2020). A T6SS was detected for the first time in the genomes of A. hydrophila and A. salmonicida (Seshadri et al., 2006; Reith et al., 2008).

Proteases are among the main virulence factors in Aeromonas (Sakai, 1985). Three types of proteases, metalloproteases, acetylcholinesterases and serineproteases are produced by Aeromonas (Seshadri et al., 2006). Both metallo- and serine proteases are secreted trough a T2SS by A. hydrophila (Sandkvist, 2001). A serine protease (AspA) as well as a glycerophospholipid:cholesterol acetyltransferase (GCAT; a lipase; see below) are secreted by A. salmonicida and were previously thought to be main virulence determinants (Buckley et al., 1982; Lee and Ellis, 1990; Whitby et al., 1992; Coleman and Whitby, 1993; Bernoth et al., 1997), however, it has later been shown that their deletion has minor effects on pathogenicity (Vipond et al., 1998).

The presence of hemolytic capability in Aeromonas is associated with the development of lesions and mortality in fish species such as rainbow trout (Ellis et al., 1988). Aeromonas species such as A. hydrophila produce α-hemolysin (HlyA), a pivotal virulence factor causing cell-rounding and apoptosis (Tekedar et al., 2019a). HlyA is an Rtx-type toxin that are known to be secreted through T1SS in Vibrio species (Boardman and Satchell, 2004). Other pore-forming Hemolysins including H-lysin, T-lysin, salmolysin, ASH1, ASH3 and ASH4 are produced by A. salmonicida to lyse erythrocytes (Titball and Munn, 1981, 1985; Nomura et al., 1988; Hirono and Aoki, 1993). Secretion mechanisms of most of the hemolysins discussed here are not yet investigated. However, mutation of a T2SS in A. hydrophila led to impaired hemolytic activity suggesting that the T2SS may be one of a secretion mechanism for at least some hemolysins in some members of the Aeromonas species (Barger et al., 2020). Aerolysin, a hemolysin, is a cytolytic pore-forming toxin produced by A. hydrophila, A. salmonicida and A. sobria (Chakraborty et al., 1990; Wilmsen et al., 1990; Hirono and Aoki, 1993; Fivaz et al., 2001) which binds to host cells and leads to increased membrane permeability. Aerolysin from A. hydrophila is known to be secreted through a T2SS (Ast et al., 2002).

Glycerophospholipid:cholesterol acetyltransferase (GCAT) is an unusual lipase involved in the pathogenicity of A. salmonicida (Lee and Ellis, 1990). Combinations of GCAT and lipopolysaccharide (LPS) from A. salmonicida have lethal effects on salmon and rainbow trout (Røsjø et al., 1993; Lachmann et al., 1998). Glycerophospholipid:cholesterol acetyltransferase is secreted through T2SS (Sandkvist, 2001).

The role of elastases from numerous bacterial pathogens as virulence factor is well documented (Kamath et al., 1998; Cascón et al., 2000; Bleves et al., 2010; Li J. et al., 2019). Among these, an extracellular elastase AhyB produced by A. hydrophila has high elastolytic activity in non-fish model and is essential for pathogenicity (Cascón et al., 2000). AhyB is secreted through T2SS (Cascón et al., 2000; Barger et al., 2020).

A DNase produced and secreted through a T2SS is present in the genome of A. hydrophila (Sandkvist, 2001), which is not yet fully characterized but is predicted to have an impact in virulence.

An extracellular ADP-ribosylating toxin (AexT) is produced by A. salmonicida, similar to the mechanism of Exoenzyme S (ExoS) in Pseudomonas aeruginosa (Yahr et al., 1996a,b) to modify host cell proteins hence induce disease. One example of this is AexT from A. salmonicida (Burr et al., 2003; Dacanay et al., 2006) where experimental evidence confirmed that this ADP-ribosylating toxin is directly translocated into the host cytosol by T3SS, similar to its P. aeruginosa homolog ExoS (Yahr et al., 1996a,b; Braun et al., 2002; Dacanay et al., 2006; Silver and Graf, 2009).

T3SSs are generally considered as a key virulence factor in many bacterial pathogens including Aeromonas due to their needle structure enabling injection of specific toxins directly into the host cytosol (Vilches et al., 2004; Dacanay et al., 2006; Coburn et al., 2007; Fast et al., 2009; Austin and Austin, 2016). Several T3SS effectors of Aeromonas are reported to affect the host immune response. AopH and AopO are effectors secreted by A. salmonicida that are secreted via the T3SS and that are highly similar to the effector proteins YopH and YopO from Yersinia species that influence cytoskeleton functions. YopH dephosphorylates tyrosines in focal adhesion proteins while YopO modulate the function of Rho GTPase to impair its roles in gene transcription, regulate actin cytoskeleton, control cell cycle and intracellular vesicle transport (Aepfelbacher, 2004). AopP is injected directly into host cells via T3SS to influence the host inflammatory response through inhibiting the nuclear factor- κB (NF-κB) signaling pathway (Fehr et al., 2006; Frey and Origgi, 2016). Other T3SS effector proteins such as the inositol polyphosphate 5-phosphatase Ati2 (Dallaire-Dufresne et al., 2013), HrpJ or BopN-like effector protein (AopN) (Nagamatsu et al., 2009; Crabill et al., 2012; Bergh and Frey, 2014), and ExsE of A. salmonicida have been shown to down-regulate host inflammatory responses (Bergh and Frey, 2014). In contrast to this, Aerolysin produced by A. hydrophila is secreted by a T2SS (Sandkvist, 2001).

The T3SS in A. salmonicida can be lost through genome modifications triggered by mobile elements such as insertion sequence (IS) elements when the bacteria are grown at temperatures exceeding 25°C, changing the strain phenotype from virulent to non-virulent (Tanaka et al., 2012, 2017). Whole genome analysis of 33 strains of A. hydrophila indicate the presence of T3SS, and mutation of two of the T3SS genes aopB (a translator) and aopD (a transmembrane protein) decreases cytotoxicity in carp epithelial cells, reduces virulence in blue gourami and increases phagocytosis (Yu et al., 2004). However, some hyper-virulent strains of A. hydrophila lack core components of the T3SS, which may indicate that these pathogens have an alternative secretion mechanism for virulence factors (Hossain et al., 2013; Pang et al., 2015).

Piscirickettsia salmonis, a member of Piscirickettsiaceae family, genus Piscirickettsia is the causative agent of piscirickettsiosis, also known as salmon rickettsial syndrome (SRS), a disease with high mortality that affects several seawater fish species. P. salmonis is non-motile, aerobic, encapsulated and pleomorphic (Fryer et al., 1990, 1992). Piscirickettsia salmonis is an intracellular pathogen causing remarkable economic loss in the aquaculture industry worldwide (Rozas and Enríquez, 2014; Figueroa et al., 2019). Diseased fish are dark in color, anemic where their kidney is swollen and the liver develops lesions (Fryer et al., 1992).

The main routes of entry of P. salmonis in rainbow trout is through skin and gills (Smith et al., 1999). Studies on P. salmonis adherence and mode of invasion using fish eggs show that the pathogen attaches to the ova by means of membrane extension which allows penetration into the cell (Larenas et al., 2003). P. salmonis replicates within membrane-bound cytoplasmic vacuoles in host cells (Fryer et al., 1992). In stress conditions, P. salmonis form a biofilm-like cell aggregate that disintegrates when treated with cellulase, which indicates the presence of polysaccharides that are common for biofilm formation (Marshall et al., 2012). In addition, some lectins show strong binding to the biofilm-like structure, which confirms the presence of an exopolysaccharide (Rozas and Enríquez, 2014).

Piscirickettsia salmonis is capable of growing and surviving in fish macrophages where the bacteria are partially enclosed into vacuole membrane vesicles which escape destruction within phagolysosomes (Almendras and Fuentealba, 1997; McCarthy et al., 2005, 2008). Piscirickettsia salmonis possess genes belonging to the Dot/Icm-type IV secretion system (T4SS) which is a type of secretion system utilized as major virulence mechanism in related intracellular pathogens such as Legionella pneumophila and Coxiella burnetii where the system is used for intracellular survival and replication (Thomas et al., 2020). It is shown that P. salmonis-containing vacuoles do not fuse with lysosomes, which suggests the presence of bacteria-mediated interference with the endosomal maturation process to ensure bacterial survival. This process depends on the Dot/Icm-type IV secretion system that delivers effector proteins into the host cytosol (Rozas and Enríquez, 2014; Zúñiga et al., 2020). As an additional virulence factor, P. salmonis contains lipopolysaccharides with endotoxin activity (Cvitanich et al., 1991). Moreover, P. salmonis-infected fish show a downregulation of genes involved in the adaptive immune response (Tacchi et al., 2011).

The molecular virulence mechanisms of P. salmonis are poorly described. However, it is known that extracellular extracts of P. salmonis contain products with cytotoxic and exotoxin effects (Rojas et al., 2013). Outer membrane vesicles containing several outer membrane proteins are released by P. salmonis during intracellular replication, including OmpA, which is involved in biofilm formation, adherence or invasion in other species. The chaperonin Hsep60, which is a cytoplasmic component but has been shown to be secreted by some pathogenic bacteria (Garduño et al., 1998; González-López et al., 2013) is presumably involved in adherence to host cell membranes (Ensgraber and Loos, 1992; Huesca et al., 1996). The chaperone HtpG that is involved in pathogenesis of E. tarda (Dang et al., 2011) is detected in the vacuoles during P. salmonis infections (Oliver et al., 2016).

Genome analysis of P. salmonis shows the presence of several other potential virulence factors (Ortiz-Severín et al., 2019). These include a Phospholipase D-like domain protein similar to the toxin Ymt from Yersinia pestis (Otsuka, 2016); three PipB2 pentapeptide repeate-containing proteins that potentially contribute to replication in intracellular vesicles as described for their homologs in Salmonella enterica (Knodler and Steele-Mortimer, 2005), an ATPase related to the flhG ATPase of Campylobacter fetus, which is part of the flagellar apparatus (Henderson et al., 2020), and a glutamate-1-semialdehyde-2,1-aminomutase whose Haemophilus somnus homolog is incolved in heme-dependent iron uptake (Villarreal, 2008).

Moritella viscosa, a member of Moritellaceae family, is a rod-shaped psychotrophic bacterium that is a main causative agent of winter ulcer, a cold season disease that infects a wide variety of salmonid fish in sea water. Diseased fish shown swelled skin, and develop lesions, hemorrhages and tissue necrosis. The mortality rate of winter ulcer is relatively low but the disease represents a significant fish welfare problem (Bruno et al., 1998; Løvoll et al., 2009).

Adherence of M. viscosa to various mucosal surface has been reported (Tunsjø et al., 2009). Gills are reported as the site of entry of M. viscosa from where the pathogens penetrate further into muscles, kidney, spleen and liver, a sign that the pathogen can cause systemic infections (Løvoll et al., 2009). The genome of M. viscosa comprises both flagella and type IV pili system genes. Adherence to fish cells is facilitated by flagella and higher level of adherence is recoded at low temperature. During adhesion by M. viscosa, aggregation of F-actin microfilaments of the cell line CHSE was observed and the host cell membrane disrupted (Tunsjø et al., 2009).

Moritella viscosa seem to survive in the host using escape mechanisms that suppress immune responses and help to evade the host immune system (Løvoll et al., 2009). Moritella viscosa contains lipopolysaccharides (LPS) and other outer membrane antigens with potential to protect the pathogen in host cells (Heidarsdottir et al., 2008). Moritella viscosa uses a metallopeptidase, MvP1 (see below for details) that is involved in invasion and dispersion of the pathogen (Bjornsdottir et al., 2009).

The virulence mechanisms of M. viscosa have not been extensively studied. The extracellular products (ECP) of the pathogen contain several secreted virulence factors with both cytotoxic and hemolytic activities that lead to the disease phenotype in Atlantic salmon (Bjornsdottir et al., 2009). The ECP contains enzymes and toxins (Benediktsdóttir and Heidarsdóttir, 2007). Genome analysis of M. viscosa shows the presence of three types of secretion systems: T1SS, T2SS and T6SS. In addition, vesicle-like structures have been observed by electro-microscopy (Tunsjø et al., 2009; Bjornsdottir et al., 2012).

The metallopeptidase MvP1 is an extracellular protease secreted by M. viscosa, formerly identified as vibrolysin with virulence related activities. By itself it is non-lethal to salmon at low concentrations. But MvP1 causes severe hemorrhages and degrades host tissues leading to necrosis, and affecting adhesion. MvP1 is suggested to be involved in invasion and dispersion in the host (Bjornsdottir et al., 2009; Björnsdóttir, 2011).

Virulence-related genes detected in several M. viscosa strains include the T6SS ATPase (clpV) and hemolysis co-regulated proteins (hcp) (Bjornsdottir et al., 2012) as well as Bacterioferritin homologs, which are known to enhance Pseudomonas putida survival in iron depleted environments (Chen et al., 2010). Further examples include a Hemagglutinin (hemG), a lectin that is produced during cell aggregation (Romeo et al., 1986) and a multifunctional autoprocessing repeats-in-toxin (martxA) from the RTX family of toxins that are secreted via a T1SS (Satchell, 2007).

Pseudomonas anguilliseptica, is a rod-shaped and flagellated member of the Pseudomonadaceae family (Palleroni, 2010). Pseudomonas anguilliseptica is a known pathogen and a serious threat to the production of a variety of fish species cultured in marine and brackish water in different parts of the world (Stewart et al., 1983; Wiklund and Bylund, 1990; Toranzo and Barja, 1993; Lönnström et al., 1994; Berthe et al., 1995; Fernández et al., 2002; Austin and Austin, 2016). The pathogen causes a disease named “red spot disease” also known as “sekiten-byo,” a hemorrhagic septicemia that was first discovered in Japan (Wakabayashi and Egusa, 1972). The disease caused by P. anguilliseptica is sometimes associated to winter disease; however, this is considered as misrepresentation as some authors consider winter disease as multifactorial condition linked with cold and stressful environments (Tort et al., 1998; Contessi et al., 2006).

Despite the seriousness of the threat of P. anguilliseptica to the aquaculture industry worldwide, knowledge on virulence mechanisms of this pathogen is scarce. The site of entry for P. anguilliseptica is not clear, but the presence of flagella indicates that the pathogen has the capacity for motility and host cell adhesion (Berthe et al., 1995). P. anguilliseptica possesses capsular (K) antigens, which was first observed in a case of eel infection. This antigen may be used for escaping complement mediated killing by fish serum (Nakai, 1985; López-Romalde et al., 2003b).