# Bacterial Type I Toxins: Folding and Membrane Interactions
## 6 Concluding Remarks and Future Perspectives  
In this review, we present an overview of data on structure and membrane interactions of toxins that belong to type I TA systems. The common features of the membrane-associated type I toxins, a small size less than 60 amino acids, an important hydrophobic character, a putative \(\alpha\)-helical transmembrane domain, and the presence of cationic residues generally localized at the C-terminal domain, allow them to directly interact with bacterial membranes that are mostly negatively charged. In most cases, the membrane-associated type I toxins display their toxicity by interfering with membrane integrity through depolarization and/or permeabilization associated with an intracellular ATP drop. Only two type I toxins, HokB and TisB, have been experimentally demonstrated as pore-forming peptides. For HokB, the pore formation is directly linked to persister cells formation, in contrast to TisB. For the other toxins acting on membrane as a primary detected effect, the mode of action remains to be determined by a global biophysical approach. If they do not form pores, the mode of action of these type I toxins may follow the model of membrane disruption via the \"carpet\" mechanism in which peptides bind to the surface of the membrane and provoke a detergent-like effect as shown for antimicrobial peptides [84]. As exceptions, four type I toxins (Fst, BsrG, AapA1, and LdrD) display their toxicity by inducing morphological changes as primary detected effect prior to membrane disruption. This suggests that these toxins may exert their toxic effect indirectly or in conjunction with other partners. Some peptides have been described as regulating degradation of membrane proteins, stabilizing P-type ATPase transporters, and modulating the activity of two-component systems [85]. The anchoring of these peptides to the membrane could facilitate interactions with targets. When comparing the sequences and structures of membrane-associated type I toxins, it is difficult to see a common mechanism of action. As an example, LdrA and LdrD toxins display a different mechanism of action despite 74% sequence homology (Figures 2 and 3) [80]. Moreover, many studies reporting type I toxin actions were based upon episomal overproduction, which is an inadequate model where the toxin concentration is not controlled and generally much higher than in reality; it could induce off-target effects that would not appear in natural conditions. It can be also noted that, in most cases, the effect of the membrane-associated type I toxins on the bacterial morphology or on the membrane integrity is usually left unexplored, as only one of these effects is studied. Like DinQ or Lpt toxins, both changes can be observed after toxin overproduction and it is tempting to speculate that some toxins may attach through both mechanisms to induce growth stasis or cell death. We aimed here at giving a chronology of the effects induced by the overproduction of toxins in order to highlight the primary effects on bacterial cells and to get insights to compare the associated mechanisms of action. Unfortunately, some studies only focused on the consequences of the overexpression at a specific time after induction and did not provide kinetic data. The information about these toxins is thus incomplete. It is also important that some experiments required an extra incubation time after sampling, like for membrane depolarization measurements, which gives a bias of the \"time after induction\" indicated in Figures 2 and 3. New hypotheses regarding the biological functions of the membrane-associated type I toxins will arise from a better understanding of their mechanisms of action and the identification of their molecular targets. Remarkably, in the case of type V _ghoT_/GhoS TA locus, the GhoT toxin is predicted to be a small membrane protein (57 amino acids) that alters membrane integrity, leading to a drop in PMF and ATP levels, thereby promoting _E. coli_ persistence [86].