## 3 Discussion
Therefore, in the absence of significant depolarization, one can infer that Pln149-PEP20 has other undiscovered mechanisms of action. Depolarization occurred in increments, likely because of the peptide mode of interaction with the membrane. The SCRD data was a way to evaluate in vitro the interaction of this new peptide with membranes. The binding of Pln149-PEP20 was also guided by electrostatic interactions with the lipid surface, especially in highly packed lipid systems. These findings agree with the higher activity of the peptide against Gram-positive bacteria. Recent data show that Plantaricin 149 acts via the carpet mode of action [10], in which the peptides interact with the membrane surface (usually with the hydrophobic part facing the membrane and the hydrophilic part facing the solvent phase). The o-SCRD for Pln149-PEP20 revealed that the helix was in contact with the lipid headgroups during the entire membrane permeation process and was not inserted across the membrane hydrophobic core, compatible with the carpet mode of action as well. Pln149-PEP20 binding might promote peptide accumulation at the lipid surface until it reaches a threshold concentration at which the membrane is efficiently disturbed, as described by the carpet-like model. Thus, depolarization increments can be explained by the SCRD data--they represent the threshold concentrations that need to be achieved in the membrane. In vitro selection showed not only the peptide's low propensity to cause resistance compared to conventional antibiotics. The assay also helped clarify the mechanism of action of Pln149-PEP20 by allowing us to investigate what kind of alterations can lead to a lower sensitivity to the molecule. It is unclear which of the mutations obtained for the in vitro-selected strains is essential for Pln149-PEP20 sensitivity reduction in _S. aureus_. Although CfIA has a highly negative region due to aspartate charges, its contribution to the cell surface charge has never been reported. CfIA and FlbPA are essential virulence proteins of _S. aureus_ that adhere to human body proteins, such as elastin, fibronectin, and fibrinogen, during the infection process [34]. The latter commonly occurs in both CfIA and FlbPA. A mutation in FlbPA occurs in the A subunit, which binds to fibrinogen. No evidence exists for the interaction of Pln149-PEP20 with fibrinogen binding sites, mainly because this molecule is highly anionic at physiological pH, unlike AMPs. Therefore, it is likely that these mutations were selected as a form of regulation because Pln149-PEP20 susceptibility decreases. Studies have already reported that MRSA has a lower expression of virulence factors when resistant to daptomycin than when sensitive to the same antibiotic. This lower expression guarantees a long and persistent infection versus an acute infection in the short term [35; 36]. The DUF1672 domain-containing protein has an important function related to Pln149-PEP20, given that a mutation occurs in two different biological replicas of the assay. Recent studies indicate that this family is a constituent of the lipoproteinome of _S. aureus_[37], and that this domain can encode lipoproteins that are added to the outer facet of the lipid bilayer. Although much remains to be characterized about these lipoproteins, they are associated with cell wall synthesis, electron transport, nutrients, and even surface stress response [38]. This finding reinforces previous results related to Pln149-PEP20's mechanism of action. The bifunctional (p)ppGpp/guanosine-3'-5'-bis(diphosphate)-3'-pyrophosphases is intrinsically related to the stringent response. They are usually synthesized in response to nutrient limitation, rRNA degradation, enzymatic activation, and replication [39]. There is a relationship between the expression of alarmones and starvation reactions due to a lack of amino acids or as a response to cell wall stresses. The transcription of the alarmones is related to tolerance to antibiotics that act on the cell wall, such as vancomycin and ampicillin.