Artificial genetic augmentation of T6SS activity affects effector repertoire and host toxicity

Abstract

ABSTRACT Type VI secretion system (T6SS), a toxic effector-delivery apparatus primarily studied for its antibacterial properties, has recently emerged as a widespread anti-eukaryotic determinant, particularly in members of the genus Vibrio. Although various anti-eukaryotic effectors have been described, it remains unknown whether effectors target distinct hosts in a similar manner. Moreover, it is unclear whether the entire effector arsenal encoded in a single genome is co-regulated across different conditions and by the same regulators. Here, we employ the anti-eukaryotic T6SS3 of Vibrio proteolyticus to address these knowledge gaps. By monitoring toxicity during infection of oyster hemocytes and comparing the results with previous infections of murine macrophages, we find that the effector Tie1 plays a role only in intoxicating murine cells, whereas Tie2 intoxicates both cell models. Furthermore, we show that artificial augmentation of T6SS3 activity by deleting the negative regulator hns1 fails to induce the orphan effector Tie3, whereas overexpression of the T6SS3-specific activator, Ats3, induces all three known effectors. Using Ats3 overexpression to activate all three effectors, we show that both Tie2 and Tie3 participate in hemocyte intoxication. Collectively, these findings indicate that analyses of T6SS-mediated infections must factor in the possibility of partial activation of the effector repertoire even when the main gene cluster is fully induced, and that the contribution of anti-eukaryotic T6SS effectors to intoxication may be host-dependent. IMPORTANCE The type VI secretion system (T6SS), a bacterial toxin-delivery apparatus, has recently been shown to mediate virulence in various marine Vibrio species. Each T6SS delivers a cocktail of toxins into a host cell, yet it is unknown whether toxins intoxicate different hosts in the same manner. Moreover, many investigations of T6SSs artificially activate the system by manipulating a transcription factor, yet it remains unknown whether different regulators induce the same toxin repertoire. Here, we show that a T6SS toxin that intoxicates one organism can be inert during the infection of another host. Furthermore, we demonstrate that T6SS-inducing regulators can activate distinct toxin repertoires. These findings suggest that conclusions about the role of T6SS toxins during infection should be drawn with caution, given the specific host model used, especially when the T6SS is artificially activated. The type VI secretion system (T6SS), a bacterial toxin-delivery apparatus, has recently been shown to mediate virulence in various marine Vibrio species. Each T6SS delivers a cocktail of toxins into a host cell, yet it is unknown whether toxins intoxicate different hosts in the same manner. Moreover, many investigations of T6SSs artificially activate the system by manipulating a transcription factor, yet it remains unknown whether different regulators induce the same toxin repertoire. Here, we show that a T6SS toxin that intoxicates one organism can be inert during the infection of another host. Furthermore, we demonstrate that T6SS-inducing regulators can activate distinct toxin repertoires. These findings suggest that conclusions about the role of T6SS toxins during infection should be drawn with caution, given the specific host model used, especially when the T6SS is artificially activated.