# 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  
For some membrane-associated type I toxins, the ectopic overexpression rapidly induces membrane depolarization and/or permeabilization, but the mechanism of action has not been clearly demonstrated by biophysical approaches. This is the case for the SprA1 (for Small pathogenicity island RNA) type I toxin (also named PepA1) of the _sprA1_/SprA1\({}_{\mathrm{AS}}\) TA system expressed in _S. aureus_ and that displays homology with the Fst/Ldr family [8; 46; 47]. SprA1 is a 30-amino acid peptide located in the membrane whose ectopic overexpression causes _S. aureus_ cell death through membrane permeabilization within 1 h upon induction (Figure 2) [23]. The _sprA1_ mRNA expression is prevented in _trans_ by the SprA1\({}_{\mathrm{AS}}\)_cis_-RNA antitoxin that base-pairs with the internal RBS and thus inhibits the toxin translation [46]. Scanning electron microscopy (SEM) shows that synthetic SprA1 provokes the lysis of competing bacteria, such as _S. aureus_ and _E. coli_, and of human erythrocytes [48]. Consequently, in contrast to Fst and AapA1 toxins [22; 24], SprA1 has an antibacterial action when added to the extracellular medium, supporting the idea that its primary toxic effect flows from its interaction with the membrane. The 3D model of the structure of SprA1 performed by NMR supports this hypothesis. SprA1 folds into an extended amphipathic transmembrane \(\alpha\)-helix that is interrupted at the P10 proline residue and slightly bends at the C15 cysteine residue in the conserved PXXXGC motif of the Fst/Ldr family [23; 49]. The N-terminus of SprA1 is unfolded and the positively charged C-terminus is folded (Table 1, Figure 1). In silico MD simulations show that, when inserted in a DPPC bilayer solvated by water molecules, the NMR SprA1 structure rapidly changes its conformation into an uninterrupted extended \(\alpha\)-helix (Figure 1) [23]. SprA1 also releases the cytoplasmic content of cells like the cationic antimicrobial peptide nisin, known to form pores leading to _S. aureus_ cell death. The authors hypothesized that SprA1 could be a pore-forming toxin [48]. Moreover, the presence of the C15 at a flexible hinge in the \(\alpha\)-helix suggests a possible dimerization that could help to form pores as shown for HokB [34]. Future biophysical studies are needed to decipher the mechanisms of action responsible for the permeabilization of both prokaryotic and eukaryotic cell membranes. The expression of SprA1 is induced upon acidic or oxidative stress in response to a drastic decrease of SprA1\({}_{\text{AS}}\) levels [23]. One hypothesis is that, after its internalization by phagocytes that triggers deleterious oxidative and acidic bursts, _S. aureus_ would induce SprA1 expression to disrupt the phagolysosome and cell membranes to escape the immune cells, kill competing bacteria, and spread into the host. Another example is the Lpt toxin, a 29-amino-acid peptide expressed from the type I TA system RNAi/RNAII identified in the plasmid DNA of _Lactobacillus rhamn_ by its sequence homology with Fst (Table 1) [50]. Induction of Lpt expression in _E. coli_ causes growth arrest, nucleoid condensation, and membrane permeabilization within 1 h upon induction (Figure 2). However, 3 h after induction, the _E. coli_ growth resumes concomitantly with the recovery of nucleoid compaction and membrane damages, highlighting that the toxin overexpression leads to cell stasis instead of cell death [51]. Both morphological modifications and membrane perturbations effects have been observed at least 1 h after induction. However, surface visualization by AFM of Lpt-expressing _E. coli_ cells evidence membrane patches linked to a disruption of the lipid bilayer. The authors suggest that Lpt acts through a detergent-like mechanism where the peptide, when added at high concentration, micelles the lipid bilayer [51].