Integrating Drug Physicochemistry and Hfq-Dependent Regulation of Ciprofloxacin Accumulation in Escherichia coli

Abstract

Ciprofloxacin (CIP), an amphoteric fluoroquinolone, undergoes pH-dependent changes in protonation state that influence membrane permeability and intracellular accumulation. Here, we investigated how drug physicochemistry and bacterial regulatory pathways jointly determine CIP accumulation in Escherichia coli using single-cell fluorescence measurements under controlled extracellular pH conditions. Mutants with hfq deletion and C-terminally truncated Hfq variants (CTR), as well as ΔompC and ΔompF mutants, were used. At pH 5, ∆ompC, ∆ompC ∆ompF, and ∆hfq strains all exhibited similarly low CIP accumulation, consistent with the limited penetration of CIP through OmpC and OmpF porins under such conditions. Importantly, we observed an increased CIP influx in ∆ctr compared to wild-type bacteria. At neutral pH 7, overall CIP accumulation increased relative to acidic conditions; however, the relative trends among the mutants remained similar. At pH 9, low efficiency of CIP accumulation occurred in the ∆ompC ∆ompF double mutant. When measuring intracellular pH, wild-type cells maintained a higher cytoplasmic pH value, ~7.4, when compared to ∆hfq, ∆ompC, and ∆ompC ∆ompF strains, which exhibited similar, lower pH values ~7.2. Atomic force microscopic studies revealed that both the Hfq-CTR and the OmpC fragment (a peptide consisting of residues 220–237) formed fibrillar structures when examined separately, but when the two peptides were combined, the resulting structures displayed an architecture distinct from that observed for either peptide alone. Together, these findings suggest a possible contribution of Hfq-modulated pH homeostasis to altered CIP accumulation.