# Bacterial Type I Toxins: Folding and Membrane Interactions
## 4 Membrane-Associated Type I Toxins Inducing Cell Morphology Changes as a Primary Detected Effect
The structural analysis performed using NMR shows that the AapA1 toxin folds into three functional domains with an amphipathic \(\alpha\)-helical transmembrane segment spanning from S9 to L28, flanked by two positively charged domains that appear to be unfolded (Table 1). The first eight amino acid residues are not required for toxicity, in contrast to the two positively charged residues, K29 and R30, at the C-terminus [24]. The addition of a SPA tag sequence at the C-terminus also suppresses the AapA1 toxicity. The C-terminal part of AapA1 is localized in the cytosol, suggesting that the K29 and R30 residues allow the binding with an intracellular target, in addition to the membrane perturbations, to interfere with cell elongation and division, leading to the formation of viable coccoids [24; 79]. Coccoids are induced by oxidative stress, probably through an increase of AapA1 expression consecutive to a drop of IsoA1 RNA level [79]. AapA1 represents an essential effector of the morphological conversion of _H. pylori_ from spirals to coccoids observed in human gastric biopsies and compared to persister cells. However, the deletion of the five homologues of AapA toxins in _H. pylori_ has no impact on the persister cells formation after oxidative stress exposure [79]. The LdrD toxin is part of the Fst/Ldr family of type I toxins and composed of 35 amino acids. It belongs to the _ldrD_/RdlD TA system located on the _E. coli_ chromosome [80]. The RdID (for Regulator detected in LDR) RNA antitoxin inhibits _ldrD_ mRNA translation, but the molecular mechanism of regulation is currently unknown. The ectopic overexpression of LdrD causes a rapid growth inhibition, loss of cell viability, and nucleoid condensation within 2 min upon induction (Figure 3) [80]. Due to the celerity of this morphological modification, the authors indicated that it is unlikely that the nucleoid condensation is linked to the accumulation of LdrD on the chromosome. The authors hypothesized that LdrD interacts with a specific cellular target important for maintaining the integrity of the nucleoid structure and the cell growth [80]. Microarray analysis showed that LdrD induction up-regulates the expression of genes involved in the purine metabolism and decreases the expression of proteins located in the membrane (Figure 3) [80]. It would be interesting to investigate the effect of the LdrD induction on the membrane permeabilization, the membrane depolarization, and the intracellular ATP level as performed for the BsrG and AapA1 type I toxins and for its homologue LdrA (57% identity and 74% homology with LdrD) to decipher the exact mechanism responsible for LdrD toxicity. NMR and CD assays showed that LdrD possesses an \(\alpha\)-helical transmembrane domain and binds to phosphocholine micelles without changing their size (Figure 1) [21; PDB id: 5 LB]. Interestingly, LdrD exhibits two conserved cationic amino acids at its C-terminal domain, which are also found for LdrD, AapA1, Fst, BsrG, and SprG1\({}_{31}\) and SprG1\({}_{44}\) toxins. Notably, they are essential for the toxicity of AapA1 toxin also inducing morphological changes as a primary observable effect (Table 1) [24]. Mutagenesis analysis could be performed to identify the amino acids that confer the toxicity of LdrD towards _E. coli_.