Cyclic di-GMP directly reprograms the multidrug resistance machinery via UspG-mediated sequestration of RamR in Klebsiella

The integration of environmental cues to counter selective pressures is crucial for the epidemiological success of major human pathogens. Klebsiella pneumoniae poses an increasing critical public health threat due to its high biofilm forming capacity and adaptive antimicrobial resistance. While the second messenger cyclic di-GMP (c-di-GMP) is a key regulator of bacterial cellular physiological adaptations, its downstream effectors that control antibiotic resistance remain unknown in K. pneumoniae. Unlike c-di-GMP metabolizing enzymes, which contain highly conserved GGDEF or EAL domains, effectors enable signal transduction through structurally heterogeneous sensing domains that defy homology-based prediction. Here, we identified the universal stress protein UspG (AVR78_17055) as a cryptic, direct c-di-GMP effector in extended-spectrum beta-lactamase (ESBL)-producing strain K. quasipneumoniae ATCC 700603. Utilizing site-directed mutagenesis and EMSA, we demonstrate that UspG senses elevated intracellular c-di-GMP levels, thereby promoting biofilm formation, via N39 and K116 residues. Mechanistically, c-di-GMP binding enhances the binding affinity of UspG to the transcriptional repressor RamR. This specific protein sequestration antagonizes RamR, a transcriptional repressor that regulates RamA expression, thereby derepressing the ramA locus and unleashing a regulatory program that fortifies lipid A biosynthesis, upregulates multidrug efflux pumps expression, and promotes biofilm development. Importantly, this c-di-GMP–UspG axis is not restricted to ESBL-producing lineages. Through mutagenesis verification, we discovered similar phenotypic dependency in hypervirulent K. pneumoniae ATCC 43816. These findings indicate that UspG is functionally conserved across Enterobacteriaceae. By elucidating how Klebsiella exploits UspG to bridge intracellular nucleotide signaling with acute environmental adaptation, our study provides a new therapeutic target for recalcitrant Klebsiella infections.Author summary: Multidrug-resistant Klebsiella pneumoniae represents a high-priority public threat challenge, driven by its capacity for robust biofilm formation and extensive antimicrobial resistance. However, the central regulatory mechanisms governing the transition of this pathogen from a susceptible to a resistant state remains to be elucidated. In this study, we discovered a previously missing molecular link in this bacterial defense strategy. We identified a universal stress protein, which we named UspG, that acts as a novel receptor for a widespread bacterial chemical messenger cyclic di-GMP in Klebsiella. Critically, our finding uncovers how bacteria utilize a conserved second-messenger system to rapidly modulate fixed genetic resistance elements—such as efflux pumps—in response to environmental cues. By bridging intracellular stress signaling with the RamA-mediated resistance regulon, this work uncovers a conserved survival mechanism in Klebsiella species that offers a strategic target for antimicrobial intervention. Unveiling this regulatory pathway not only advances our understanding of bacterial adaptation but also reveals a vulnerable target for designing innovative therapies against this priority pathogen in clinical environments.