Mechanisms of cytotoxicity induced by Vibrio anguillarum, a bacterial pathogen of fish

Many bacterial pathogens are capable of producing potent cytotoxins that can hinder the host cells in a variety of ways. In many cases, the toxins eventually result in the death of the cell. While the death of the cell is certain, the pathway that the cell takes to reach that end point can vary. Historically, cell death was described as a programmed cell death (apoptosis) or an accidental cell death (necrosis). Recent research however has shown that cells have a much more variable procedure when it comes to their eventual demise. Of these pathways, the apoptotic pathway has been characterized the best. As such, there are multiple experimental methods for determining host cell death, most of which are aimed at determining if host cells exhibit classical apoptotic markers. Several cell death assays can be used in conjunction with one another, allowing for an accurate description of how a toxin affects its host cell. Vibrio anguillarum, the causative agent of warm-water vibriosis in fish, possesses several virulence factors, including two gene clusters (the vah1 and the rtx gene clusters) that encode for hemolytic activity. Using Atlantic salmon kidney (ASK) cells, we have previously shown that the toxins produced by these gene clusters, Vah1 and RtxA, cause different morphological responses. Vah1 causes excessive vacuolation in ASK cells and treatment with RtxA causes ASK cells to round and die. While these activities show that both Vah1 and RtxA are cytotoxic, it was unknown whether these cytotoxins induced apoptosis or necrosis in ASK cells. To determine this, the current study tested the reaction of ASK cells treated with live V. anguillarum strains using light microscopy, membrane integrity assays, and caspase-3/-7 activation assays. We found that when ASK cells are treated with wild type V. anguillarum at a low MOI (MOI = 20), ASK cells shrink, round up, and exhibit some membrane blebbing. These morphological changes were lost when cells were treated with V. anguillarum strains that lacked the RtxA toxin. The V. anguillarum strain which lacked the RtxA cysteine protease domain (CPD) caused ASK cells to remain the same size or slightly swell, while also producing some membrane blebbing. After testing for membrane permeability, we observed that ASK cells treated with wild type V. anguillarum cells in high numbers (MOI = 200) showed ∼40% lysis after being treated for 4 h, with little to no lysis being observed until 2 h of treatment. ASK cells treated with RtxA-deficient strains showed little activity, while those with a functional RtxA protein showed values similar to the wild type. The strain that contained the CPD deletion of RtxA was shown to induce lysis at approximately wild type levels at these high levels of infection. When ASK cells were subjected to low infections (MOI = 20), little to no membrane permeability was observed after 4 h of treatment. The wild type strain of V. anguillarum induced only 10–15% lysis after the treatment, with similar levels seen in both the CPD mutant and RtxA-only producing strains. Finally, we observed that ASK cells treated with lower MOI values (MOI = 20 and 50) of V. anguillarum wild type cells induced better caspase-3/-7 activity. Furthermore, V. anguillarum strains that contained a fully functional RtxA induced caspase activity, while those with an altered toxin did not. Taken together, the data showing ASK cell shrinkage and blebbing, the low levels at which ASK cells exhibited the loss of membrane integrity, and the activation of caspase during treatment with the wild type V. anguillarum strongly suggest that V. anguillarum cells induce apoptotic cell death. Additionally, the data also suggest that apoptotic death is induced primarily by the V. anguillarum RtxA toxin. Further, cleavage of the individual effector domains within RtxA by the CPD is needed to induce the apoptotic cascade