T3SS effectors in Vibrios: Homology in sequence, diversity in biological functions?

Vibrio alginolyticus is a Gram-negative bacterium that thrives in marine environments, and is also an important pathogen that causes vibriosis in a variety of marine animals. Infections in cultivated fish and invertebrates result in significant financial losses for aquaculture industries worldwide [1-3]. Furthermore, this bacterium can also be an opportunistic pathogen for humans, causing diarrhea as well as extraintestinal infections such as otitis and wound infections [4-6]. The mechanism of pathogenesis of this bacterium has not been completely elucidated but a number of virulence factors have been described, including an alkaline serine protease [7-8], hemolysins [9], siderophores used as host iron scavengers [10] and a type III secretion system [11]. The type III secretion system (T3SS) is a conserved apparatus among several Gram-negative bacteria that delivers effector proteins directly into host cells [12]. Effector proteins can behave as virulence factors that manipulate host cell physiology and cause a diversity of cellular responses and cell damage. T3SS have been characterized in a number of Vibrionaceae species. The first report of a T3SS in Vibrio was in V. parahaemolyticus where two sets of T3SS gene clusters, T3SS1 and T3SS2, were discovered [13]. T3SS1 is found in all V. parahaemolyticus strains, while T3SS2 is present only in KP-positive strains. Although initially thought to be absent from V. cholerae T3SS gene clusters were subsequently reported in non-O1, non-O139 strains [14,15] and in non-toxigenic O1 V. cholerae [16]. Similar clusters were identified in other Vibrio species, including V. alginolyticus [11,17], V. mimicus [18], V. harveyi [19] and V. tubiashii [17], among others. Recently, a T3SS was also reported in the type strain of another member of the Vibrionaceae family: the fish and human pathogen Photobacterium damselae subsp. damselae [20]. The best studied Vibrio T3SS so far is T3SS1 from V. parahaemolyticus, a pathogen for humans as well as for marine animals. This system induces death of mammalian cell lines in a multifaceted process that starts with autophagy, followed by cell rounding, and culminating with cell death in a caspase-independent process [21,22]. The effector protein VopQ is necessary and sufficient to induce autophagy in HeLa cells through its interaction with the Vo domain of the V-type H -ATPase in lysosomal membranes [23,24]. Another effector, VopS, is involved in cell rounding and in the collapse of the actin cytoskeleton by inhibiting Rho GTPases [25]. The V. alginolyticus genome encodes a system homologous to the V. parahaemolyticus T3SS1, including homologues of VopQ and VopS. Interestingly, it was shown that the V. alginolyticus T3SS was responsible for causing rapid apoptosis, cell rounding and osmotic lysis in the fish cell line EPC [11]. These findings were in clear contrast with the effect of V. parahaemolyticus on mammalian cells which is characterized by the activation of autophagy but not of apoptosis [21,22]. Interestingly, when the same V. alginolyticus strain was used to infect the human HeLa cell line apoptosis was not induced but activation of autophagy occurred instead [26]. In the current issue of Virulence, Zhe Zhao and colleagues [27] uncovered the role of two V. alginolyticus T3SS effector proteins, Val1686 and Val1680, in apoptosis, cell rounding and osmotic lysis in the fish cell line FHM. The authors report the singularities of these two V. alginolyticus proteins, which are homologues of the V. parahaemolyticus T3SS1 VopS (91% id.) and VopQ (88% id.) effectors, respectively. Zhao and colleagues show here that V. alginolyticus VopS not only contributes to cell rounding of fish cells by inhibiting Rho GTPases but that it also is essential for the induction of

Vibrio alginolyticus is a Gram-negative bacterium that thrives in marine environments, and is also an important pathogen that causes vibriosis in a variety of marine animals. Infections in cultivated fish and invertebrates result in significant financial losses for aquaculture industries worldwide [1][2][3]. Furthermore, this bacterium can also be an opportunistic pathogen for humans, causing diarrhea as well as extraintestinal infections such as otitis and wound infections [4][5][6]. The mechanism of pathogenesis of this bacterium has not been completely elucidated but a number of virulence factors have been described, including an alkaline serine protease [7][8], hemolysins [9], siderophores used as host iron scavengers [10] and a type III secretion system [11].
The type III secretion system (T3SS) is a conserved apparatus among several Gram-negative bacteria that delivers effector proteins directly into host cells [12]. Effector proteins can behave as virulence factors that manipulate host cell physiology and cause a diversity of cellular responses and cell damage. T3SS have been characterized in a number of Vibrionaceae species. The first report of a T3SS in Vibrio was in V. parahaemolyticus where two sets of T3SS gene clusters, T3SS1 and T3SS2, were discovered [13]. T3SS1 is found in all V. parahaemolyticus strains, while T3SS2 is present only in KP-positive strains. Although initially thought to be absent from V. cholerae T3SS gene clusters were subsequently reported in non-O1, non-O139 strains [14,15] and in non-toxigenic O1 V. cholerae [16]. Similar clusters were identified in other Vibrio species, including V. alginolyticus [11,17], V. mimicus [18], V. harveyi [19] and V. tubiashii [17], among others. Recently, a T3SS was also reported in the type strain of another member of the Vibrionaceae family: the fish and human pathogen Photobacterium damselae subsp. damselae [20].
The best studied Vibrio T3SS so far is T3SS1 from V. parahaemolyticus, a pathogen for humans as well as for marine animals. This system induces death of mammalian cell lines in a multifaceted process that starts with autophagy, followed by cell rounding, and culminating with cell death in a caspase-independent process [21,22]. The effector protein VopQ is necessary and sufficient to induce autophagy in HeLa cells through its interaction with the V o domain of the V-type H + -ATPase in lysosomal membranes [23,24]. Another effector, VopS, is involved in cell rounding and in the collapse of the actin cytoskeleton by inhibiting Rho GTPases [25].
The V. alginolyticus genome encodes a system homologous to the V. parahaemolyticus T3SS1, including homologues of VopQ and VopS. Interestingly, it was shown that the V. alginolyticus T3SS was responsible for causing rapid apoptosis, cell rounding and osmotic lysis in the fish cell line EPC [11]. These findings were in clear contrast with the effect of V. parahaemolyticus on mammalian cells which is characterized by the activation of autophagy but not of apoptosis [21,22]. Interestingly, when the same V. alginolyticus strain was used to infect the human HeLa cell line apoptosis was not induced but activation of autophagy occurred instead [26].
In the current issue of Virulence, Zhe Zhao and colleagues [27] uncovered the role of two V. alginolyticus T3SS effector proteins, Val1686 and Val1680, in apoptosis, cell rounding and osmotic lysis in the fish cell line FHM. The authors report the singularities of these two V. alginolyticus proteins, which are homologues of the V. parahaemolyticus T3SS1 VopS (91% id.) and VopQ (88% id.) effectors, respectively. Zhao and colleagues show here that V. alginolyticus VopS not only contributes to cell rounding of fish cells by inhibiting Rho GTPases but that it also is essential for the induction of apoptosis. A deletion mutant unable to produce VopS is severely impaired in its ability to induce these two cell responses and transfection of VopS into the fish cell line FHM is sufficient to cause cell rounding and apoptosis. The authors also show that the V. alginolyticus VopQ contributes to fish cell lysis but does not induce autophagy, a characteristic that distinguishes it from its V. parahaemolyticus homologue VopQ.
The findings reported by Zhao and colleagues in the present issue of Virulence provide new insights into the mechanisms of V. alginolyticus pathogenesis for fish cells. These data unequivocally demonstrate that this marine pathogen is capable of inducing apoptosis in fish cells and this effect is dependent on the T3SS effector VopS. Importantly, these results suggest that the type of host cell line used in the experiments is a variable of the maximal importance when drawing conclusions about the biological effects caused by a T3SS effector protein.
The inherent differences between fish and mammalian cells may constitute the main reason why the V. alginolyticus and V. parahaemolyticus VopS and VopQ proteins induce distinct cellular responses. Very elegant experiments have contributed to unveil the roles of V. parahaemolyticus VopS and VopQ using mammalian cell lines and yeasts as host cell models [24,25,28,29], but information on the effects of these effectors in fish cells is scarce. In the light of the novel findings reported by Zhao and colleagues [27], it would be of high interest to compare the cellular effects caused by pairs of homologous T3SS effector proteins from different Vibrio species using the same host cell line. Future studies are needed in order to determine whether the different cellular responses elicited by V. alginolyticus and V. parahaemolyticus T3SS effectors are due to amino acid substitutions in homologous proteins or to inherent differences between fish and mammalian cells.

Disclosure of potential conflicts of interest
No potential conflicts of interest were disclosed.

Funding
This work was supported by grant AGL2016-79738-R (AEI/ FEDER, EU) from the State Agency for Research (AEI) of Spain, and co-funded by the FEDER Programme from the European Union. The support of Xunta de Galicia (Spain) with grant GRC-2014/007 is also acknowledged.