Revisiting the LuxS/AI-2–SdiA Regulatory Network in Klebsiella pneumoniae: Context-Dependent Modulation by Halogenated Thiolactones

Quorum sensing (QS) represents a promising target for anti-virulence therapy; however, effective pharmacological intervention requires a detailed understanding of regulatory network architecture and environmental context. In Klebsiella pneumoniae, the orphan LuxR-type receptor SdiA lacks a cognate LuxI synthase and instead detects exogenous acyl-homoserine lactones (AHLs), positioning it as an inter-species signal integrator. Here, we demonstrate that SdiA functions as a context-dependent regulator whose impact on biofilm formation and virulence gene expression is gated by environmental AHL availability. Using isogenic ΔluxS, ΔsdiA, and ΔluxSΔsdiA mutants in a clinical bloodstream isolate, we show that under AHL-limited conditions, SdiA promotes baseline biofilm development, whereas in the presence of exogenous C6-HSL, it restrains excessive biofilm maturation. Two-way ANOVA confirmed significant genotype, treatment, and interaction effects, establishing that SdiA-mediated regulation is signal contingent. We further investigated the halogenated thiolactone meta-bromo-thiolactone (mBTL), previously described as a QS inhibitor in Pseudomonas aeruginosa. In K. pneumoniae, mBTL acts as a context-selective modulator rather than a simple inhibitor. Under AHL-limited conditions, mBTL phenocopied ΔsdiA, reducing biofilm formation and inducing overlapping transcriptional profiles. In contrast, under AHL-replete conditions, mBTL opposed SdiA-dependent gene expression, consistent with competitive antagonism of ligand-bound receptor. RNA-seq analysis revealed substantial concordance between ΔsdiA and WT + mBTL under AHL-free conditions, with the inversion of transcriptional directionality in the presence of C6-HSL. The findings redefine SdiA as a conditional quorum-sensing integrator and identify mBTL as a ligand-context-dependent modulator of LuxR-type signaling. Our results highlight the necessity of evaluating anti-virulence compounds across relevant signal environments and introduce receptor state-selective modulation as a strategic framework for targeting hybrid quorum-sensing systems in polymicrobial pathogens.