Time-resolved transcriptomic analysis reveals envelope stress-mediated adaptation of

Gram-negative bacteria are a major cause of antibiotic-resistant infections due to their highly impermeable cell envelope which contains lipopolysaccharide (LPS) and prevents antibiotics from entering the cell. LPS biogenesis relies on the activity of the essential LPS transport machinery (Lpt), which represents an attractive and validated target for antibiotic development. The antimicrobial peptide thanatin impairs LPS transport by disrupting the assembly of the Lpt protein machinery, resulting in cell death. However, thanatin is not a suitable drug candidate due to its poor drug-like properties and the rapid emergence of resistance. Therefore, understanding the mechanisms underlying cell resistance and adaptation to thanatin is crucial for developing novel antimicrobial strategies. Here we performed RNA-seq-based kinetic transcriptomic analysis to investigate how bacterial cells respond to thanatin over time followed by a functional validation. We found that thanatin treatment induces a rapid activation of the Cpx envelope stress response system, followed by activation of Rcs phosphorelay and downregulation of the FlhDC regulon. Cpx regulated genes showed enrichment for pathways involved in cationic peptide resistance, stress regulation, protein folding, and peptidoglycan remodelling. Rcs upregulated genes were associated with capsule synthesis and biofilm formation. Finally, late response to thanatin suggests the involvement of phospholipid metabolism modulation and trafficking as a strategy to restore OM asymmetry. Overall, our results delineate the adaptive mechanisms employed by bacterial cells to counteract thanatin-induced stress through an interplay of multiple regulatory pathways controlling envelope integrity, stress responses, motility and biofilm formation.