# Bacterial Type I Toxins: Folding and Membrane Interactions
## 3 Membrane-Associated Type I Toxins Inducing Membrane Perturbations as a Primary Detected Effect
### Membrane-Associated Type I Toxins Inducing Membrane Depolarization and/or Permeability
Despite these observations, it is not excluded that cell morphology changes induced by Lpt overexpression could be the primary triggers of its toxic effect. The membrane localization of Lpt was evidenced by the fusion of the red fluorescent protein m-Cherry to the C-terminus of the peptide. This fusion or the removal of the hydrophilic C-terminal region abolishes the toxicity of Lpt, supporting the importance of the C-terminal domain for toxicity. Moreover, the substitution of a proline (P11) by a charged glutamic acid in the \(\alpha\)-helical transmembrane domain also suppresses the toxicity of Lpt, suggesting that the distortion of the \(\alpha\)-helix induced by P11 is essential for the interaction with the membrane and thus, for the toxicity, as this is the case for Fst [26]. Although Lpt, Fst, and SprA1 all share the conserved motif APXXXGXXX, where X represents hydrophobic amino acids (Table 1) [23; 52], it seems difficult to predict a similar mechanism of action for these three toxins. Indeed, the SprA1 homologue, SprA2 (also named PepA2), that displayed the conserved PXXXGC motif, does not lyse bacterial cells extracellularly but has a more hemolytic effect than SprA1 [13]. Lpt induces cell morphology changes and it is not excluded that this could be the primary trigger of its toxic effect. For this TA system, the molecular mechanisms involved in the regulation of Lpt expression by the RNA II antitoxin are currently unknown. Five other type I toxins expressed by _E. coli_ (namely ZorO, IbSc, ShoB, LdrA, and DinQ) rapidly cause membrane depolarization and/or permeabilization and/or intracellular ATP drop when overexpressed. The ZorO type I toxin was identified by bioinformatics with search parameters including tandemly duplicated, small, hydrophobic proteins by the Fozo group [8]. ZorO (for _Z_-protein often repeated) is a 29-amino-acid peptide (Table 1) whose expression is tightly repressed by a 5'-UTR secondary structure that sequesters the RBS of _zorO_ mRNA and by the OrzO (for Overexpression reduces \(Z\) protein toxicity) RNA antitoxin that base pairs to the EAP (for Exposed After Processing) region located upstream of the RBS of _zorO_ mRNA [11; 53]. When overexpressed in _E. coli_, ZorO localizes at the inner membrane and induces membrane depolarization associated with a reduction of the intracellular ATP level and an increase of ROS production, leading to the membrane permeabilization and finally to cell death within 30 min (Figure 2) [8; 54]. A mutagenesis analysis on ZorO demonstrated that only the five amino acids \"ALLRL\" spanning positions 20 to 24 are necessary for toxicity while the 13 N-terminal amino acids are dispensable [54]. Conversely to full-length ZorO, the ALLRL peptide, when added to the extracellular medium, exhibits antimicrobial activity against the Gram-positive bacteria _S. aureus_ and _B. subtilis_ and against the fungus _C. albicans_ through a mechanism of membrane permeabilization. However, it does not inhibit _E. coli_ growth, suggesting that the outer membrane of Gram-negative bacteria prevents its binding to the inner membrane [54]. These observations indicate that accessibility to the inner membrane is essential for ZorO to exert its toxicity against _E. coli._  
The IbSc (for Induction brings stasis) and ShoB (for Short hydrophobic ORF) type I toxins from the _ibSc_/SibC and _shoB_/OhsC TA systems were discovered in _E. coli_ by a bioinformatics analysis designed to identify new TA systems [55]. IbsC and ShoB are, respectively, 19- and 26-amino-acid peptides whose expression is prevented by SibC (for Short intergenic abundant sequence) and OhSc (for Oppression of hydrophobic ORF by sRNA) RNA antitoxins [56]. SibC interacts with the _libsC_ mRNA through the TRD1 (for Target Recognition Domain) and TRD2 domains to repress IbsC toxicity [57].