Investigation of cationic antimicrobial peptide stress response in Escherichia coli

Bacteria possess diverse mechanisms that allow them to sense and respond to environmental stressors, activating signaling pathways that induce specific transcriptional programs. While these stress responses are typically recognized as adaptive regulatory systems, they can also produce secondary effects on important cellular processes. In Escherichia coli, exposure to stressors, such as the presence of cationic antimicrobial peptides (cAMPs), strongly activates the PhoQ/PhoP two-component system, resulting in transcriptional upregulation of the enzyme QueE. Elevated levels of QueE lead to its co-localization with the cell division protein FtsZ at the septum, inhibiting cell division and promoting filamentous growth. However, the mechanism by which QueE-mediated septation inhibition occurs and how different stressors modulate PhoQP activation remains undefined. In this thesis, I investigate the activation of the PhoQ/PhoP system and its associated phenotypes in response to multiple cationic antimicrobial peptides, including derivatives of the human innate immune peptide LL-37: KR-12, the smallest active fragment of LL-37; RI-10, a non-toxic fragment; as well as HNP-1 and HBD-3. Using single-cell fluorescence transcriptional reporters combined with phase-contrast and fluorescence microscopy, I show that sublethal concentrations of LL-37 and its derivatives induce significant cell elongation, a phenotype dependent on PhoQ and mediated by the upregulation of QueE, which interferes with bacterial cell division. Contrary to the previous model, which suggested that peptides activate PhoQ passively by displacing its inhibitor MgrB, I observed enhanced cell elongation in ΔmgrB strains across all tested peptides, including RI-10, which lacks antibacterial activity. Our findings suggest cationic peptides such as LL-37 and its derivatives actively stimulate PhoQ through a mechanism independent of MgrB dissociation, providing an updated model for peptide sensing and signaling through the PhoQ/PhoP system. To investigate the mechanism of cell elongation via QueE, I performed mechanistic studies using fluorescently tagged cell division proteins under conditions of PhoQ/PhoP activation. These experiments reveal that QueE does not impair early divisome assembly or Z-ring formation but instead blocks septation by interfering with the recruitment of late-stage divisome proteins. Together with the other findings of cell division proteins that genetically suppress PhoQ/PhoP-dependent QueE-mediated filamentation, my work provides a better understanding of how QueE-mediated filamentation occurs in E. coli cells under AMP stress. Overall, this work establishes a mechanistic link between AMP-induced activation of PhoQ/PhoP signaling, leading to a morphological response involving the inhibition of bacterial cell division, expanding our knowledge of how stress signaling pathways interact with important cellular processes.