# Bacterial Type I Toxins: Folding and Membrane Interactions
## 2 Overview of the Membrane-Associated Type I Toxins across the Bacterial Species  
The membrane-associated type I toxins have only been described in Proteobacteria and Firmicutes [14]. We have classified the membrane-associated type I toxins whose structure and/or mechanism of action have been deciphered (Table 1). These small hydrophobic peptides like phage holins [8] or cationic antimicrobial peptides [18] all display an \(\alpha\)-helicaltransmembrane domain but have a strong diversity in length and amino acid sequence. The \(\alpha\)-helical structure was experimentally validated for some of them such as TiSb, IbSc, and LdrD in _Escherichia coli_[19; 20; 21], Fst in _Enterococcus faecalis_[22], SprA1 and SprG1 in _S. aureus_ ([23] and in this study), and AapA1 in _Helicobacter pylori_[24] (Figure 1). For the other type I toxins, we could predict the \(\alpha\)-helical transmembrane domain using the TMPRED algorithm along with the orientation of the computed in silico model of \(\alpha\)-helix from the inside (I) to the outside (O) of the bacterial membrane with a given probability ranging from ++ to \(-\) in decreasing order ([[https://embnet.vital-it.ch/software/TMPRED_form.html](https://embnet.vital-it.ch/software/TMPRED_form.html)]([https://embnet.vital-it.ch/software/TMPRED_form.html](https://embnet.vital-it.ch/software/TMPRED_form.html)), accessed on 5 April 2021) (Table 1). Among the common features, the C-terminal part is mainly predicted to be localized in the cytosol, except for the ShoB, HokB, SprA1, and SprG1 toxins, and has positively charged residues, except for IbSc, ShoB, and DinQ (Table 1). These positively charged residues were shown to be crucial for the binding/anchoring of peptides on lipid bilayers and may help interactions with the bacterial membrane that is mostly negatively charged [25]. The presence of cysteine residues in HokB, ShoB, AapA1, and SprA1 suggests a possible dimerization/oligomerization of these toxins, leading to pore formation. Sequence requirements for toxicity have been demonstrated by mutagenesis approaches for the Fst, IbSc, and AapA1 toxins [20; 24; 26]. These studies revealed that only a few residues are critical for toxicity, but the lack of sequence conservation within the type I toxins impedes the prediction of amino acids essential for toxicity by homology searches using bioinformatics tools [8]. Despite several common features, the membrane-associated type I toxins exert distinct mechanisms of action based on toxin ectopic overexpression. We can distinguish type I toxins whose overexpression induces bacterial membrane alterations as primary detected and surely toxic effects such as membrane depolarization and/or permeabilization, and the type I toxins whose overexpression induces morphological changes in bacteria as a primary detected effect prior to membrane perturbations.  
Figure 1: Overview of the structurally determined membrane-associated type I toxins. Hydrophobic amino acids are represented in gray, polar amino acids in green, positively charged amino acids in blue, negatively charged amino acids in red, and cysteines in yellow.