Two-Component Response Regulator OmpR Regulates Mucoviscosity Through Energy Metabolism in Klebsiella Pneumoniae

Hypermucoviscosity is a hallmark of hypervirulent Klebsiella pneumoniae (hvKP). However, the molecular basis of its regulation is largely unknown. We hypothesize that hypermucoviscosity is modulated via two-component signal transduction systems (TCSs). In-frame deletion mutants of all 33 response regulators of hvKP ATCC43816 were generated using CRISPR/CAS and evaluated for their impacts on hypermucoviscosity. The response regulator OmpR is required for hypermucoviscosity and virulence in a mouse pneumonia model. The Δ mutant lost its mucoidy but retained its capsule level and comparable expression, so transcriptomic analysis by RNA-Seq was performed to identify differentially expressed genes (DEGs) in Δ mutant. The top 20 Gene Ontology terms of 273 DEGs belong to purine ribonucleotide triphosphate biosynthetic and metabolic process, transmembrane transport, and amino acid metabolism. Among the overexpressed genes in the Δ mutant, the operon encoding F-type ATP synthase and the encoding glycine cleavage system were characterized further as overexpression of either operon reduced the mucoviscosity and increased the production of ATP. Furthermore, OmpR directly bound the promoter region of the operon, not the , suggesting that OmpR regulates the expression of the operon directly and indirectly. Hence, the loss of OmpR led to the overexpression of F-type ATP synthase and glycine cleavage system, which altered the energetic status of Δ cells and contributed to the subsequent reduction in the mucoviscosity. Our study has uncovered a previously unknown regulation of bacterial metabolism by OmpR and its influence on hypermucoviscosity. Hypermucoviscosity is a critical virulent factor for Klebsiella pneumoniae infections, and its regulation remains poorly understood at the molecular level. This study aims to address this knowledge gap by investigating the role of response regulators in mediating hypermucoviscosity in K. pneumoniae. We screened 33 response regulators and found that OmpR is essential for hypermucoviscosity and virulence of K. pneumoniae in a mouse pneumonia model. Transcriptomic analysis uncovered that genes involved in energy production and metabolism are highly upregulated in the Δ mutant, suggesting a potential link between bacterial energy status and hypermucoviscosity. Overexpression of those genes increased production of ATP and reduced mucoviscosity, recapitulating the Δ mutant phenotype. Our findings provide new insights into the regulation of K. pneumoniae hypermucoviscosity by a two-component signal transduction system, highlighting the previously unknown role of OmpR in regulating bacterial energy status and its influence on hypermucoviscosity.