# OxyR contributes to the oxidative stress capacity and virulence of hypervirulent Klebsiella pneumoniae ATCC 43816

## TL;DR

This study shows that the OxyR protein helps hypervirulent Klebsiella pneumoniae resist oxidative stress and cause severe infections.

## Contribution

The study identifies OxyR as a key regulator of oxidative stress resistance and virulence in hvKP through genetic and transcriptomic analyses.

## Key findings

- OxyR deletion increases susceptibility to hydrogen peroxide and reduces biofilm formation in Klebsiella pneumoniae.
- The OxyR mutant shows reduced virulence in animal models, with improved host survival and lower bacterial burdens.
- OxyR regulates genes like hemH, grxA, and katG in response to oxidative stress.

## Abstract

Hypervirulent Klebsiella pneumoniae (hvKP) is an emerging pathogen associated with severe invasive infections and high mortality, in which resistance to host-derived reactive oxygen species (ROS) is critical for immune evasion and persistence. However, the mechanisms underlying oxidative stress resistance in hvKP remain poorly understood, and the role of the global regulator OxyR in this species has not been fully elucidated.

In this study, VK055_RS16305 was first identified as an OxyR homologue in K. pneumoniae ATCC 43816 by sequence alignment. The oxyR deletion mutant was generated using a CRISPR/Cas9-based genome editing system, whereas the complemented strain was obtained using the pSTV28 plasmid carrying oxyR. We then compared their growth characteristics, susceptibility to H₂O₂, biofilm formation, and virulence in Galleria mellonella and mouse infection models, and performed RNA sequencing followed by qRT-PCR to characterize the OxyR regulon under oxidative stress.

Deletion of oxyR did not alter bacterial growth or colony morphology under non-stress conditions, but markedly increased susceptibility to H₂O₂ and impaired biofilm formation. In vivo, the oxyR mutant exhibited attenuated virulence, with improved survival of Galleria mellonella and mice and significantly reduced bacterial burdens in blood, liver, lung, and spleen, all of which were restored by genetic complementation. Transcriptomic analysis revealed that OxyR positively regulates multiple oxidative stress–associated genes, including hemH, grxA, gsk, katG, and ahpC, in response to H₂O₂.

Together, these findings demonstrate that OxyR is a key regulator of oxidative stress defense, biofilm formation, and systemic virulence in hvKP, providing new insight into OxyR-mediated pathogenic mechanisms in K. pneumoniae.