Abstract
ABSTRACT
Biofilm formation by
Helicobacter pylori
is a major driver of antibiotic tolerance and treatment failure, yet the signaling pathways that trigger biofilm development under sub-inhibitory antibiotic pressure remain poorly understood. Here, we show that sub-MIC levels of metronidazole, amoxicillin, and ciprofloxacin potently induce dense
H. pylori
biofilms. Through transcriptomic and genetic analyses, we identify the two-component system CrdRS as a central signaling hub that orchestrates this response via the bidirectional regulation of two ATP-binding cassette (ABC) transporters. Phosphorylated CrdR binds to AC-rich promoter motifs to directly activate
plpA
, which encodes a substrate-binding protein that drives exopolysaccharide secretion and matrix assembly. Concomitantly, CrdR represses
glnP
, which encodes an inner-membrane permease, thereby relieving transcriptional inhibition of the L-asparaginase gene
ansB
. This derepression triggers aberrant reactive oxygen species (ROS) accumulation, which promotes oxidative stress-dependent biofilm maturation. Through phenotypic analysis of c
rdRS
deletion mutants, phosphorylation-defective point mutants (CrdR
D53A
and CrdS
H173A
), and exogenous hydrogen peroxide (H₂O₂) induction, we demonstrate that CrdRS is required for mediating antibiotic-induced stress responses in a manner genetically separable from ROS sensing. Collectively, our findings establish a dual-mechanism model in which CrdRS orchestrates antibiotic-induced biofilm formation by simultaneously controlling matrix production and intracellular ROS generation. Notably,
glnP
expression was significantly lower in clinical multidrug-resistant isolates than in drug-sensitive ones, whereas
plpA
showed the opposite trend. These findings provide a mechanistic foundation for developing CrdRS-targeted strategies to combat biofilm-associated
H. pylori
infections.