# Bacterial Type I Toxins: Folding and Membrane Interactions
## 4 Membrane-Associated Type I Toxins Inducing Cell Morphology Changes as a Primary Detected Effect  
Some membrane-associated type I toxins affect bacterial inner membranes, but cell morphology changes are induced as primary detected effects prior to membrane damages. This class of type I toxins is represented by Fst, BsrG, AapA1, and LdrD toxins and probably by Lpt and/or DinQ toxins as discussed before. The Fst (for faecalis plasmid stabilizing toxin) type I toxin is a 33-amino-acid peptide that belongs to the RNAi/RNAII PSK TA system, expressed from the _par_ locus of the _Enterococcus faecalis_ plasmid pAD1 [65]. The [PERSON] group demonstrated that, in _E. faecalis_ and in the presence of the pAD1 plasmid, the interaction between RNAi, the messenger RNA encoding Fst, and the antitoxin RNAII is initiated at the loop of the intrinsic terminators and that it spreads to the DRa (for DNA direct repeats) and DRb sequences located at the 5' end of both RNAs. This results in a sequestration of the GUG initiation codon of RNAi and subsequently to the inhibition of Fst translation [66; 67]. The group also showed that RNAi SD sequence is sequestered within an intramolecular stem-loop and that translation repression is maintained until interaction with RNAi [68; 69]. These studies also demonstrated that, upon pAD1 plasmid loss, RNAII, in complex with RNAi, is degraded by RNases and that Fst is produced from RNAi, resulting in cell death. Indeed, ectopic overexpression of Fst rapidly provokes a chromosome segregation defect evidenced by nucleoid condensation and misplaced septa, resulting in daughter cells with little or without DNA within 15 min after induction in _E. faecalis_ (Figure 3) [70]. These cell division abnormalities precede the simultaneous inhibition of DNA replication, transcription, and translation, membrane permeabilization, and thus cell growth nearly 45 min after Fst induction, highlighting that morphological changes are the primary detected effects related to the toxicity of the toxin (Figure 3) [71]. Moreover, Fst overexpression did not lead to the formation of \"ghost\" cells or leakage of cellular content as for HokB and TisB toxins [9; 30; 71], confirming that Fst is not a pore-forming toxin. Synthetic Fst has no effect on bacteria, fungal cells, or erythrocytes when added to the extracellular medium [22; 71], indicating that Fst is localized within the membrane to facilitate interactions with a specific membrane-bound intracellular target or is intracellularly modified rather than being directed against the membrane itself as observed with antimicrobial peptides. Interestingly, microarray analysis showed that Fst overexpression increases the expression of several membrane transporters 1 h after induction. This expression is detrimental for the bacteria potentially by depleting the pool of intracellular ATP and/or by perturbing the membrane integrity secondary to defects on nucleoid structure, chromosome segregation, and cell division (Figure 3) [72]. CD, NMR, and MD simulation experiments demonstrated that Fst forms an \(\alpha\)-helical transmembrane structure between amino acids 4 and 26 with a slight bend at the G15 and a disordered charged C-terminal domain (Table 1). The presence of negatively charged residues (D26 to D30) at the C-terminal domain suggests that Fst binds to the cell membrane with the C-terminus pointing into the cytosol since the outer side of Gram-positive membranes is negatively charged (Figure 1) [22]. The authors hypothesized that Fst anchoring at the membrane facilitates binding with membrane-bound target through the C-terminal domain, which would become structured after binding [22]. However, mutagenesis analysis demonstrated that the Fst C-terminal domain is dispensable for the toxin toxicity, although its deletion seems to reduce toxicity [49]. Conversely, the hydrophobic domain, notably the P11 and the G15 residues present in the conserved motif APXXXGXX, and the two charged amino acids K2 and D3 at the N-terminus, are essential for the toxicity of Fst [26].