# The IrlS2-IrlR2 two-component system is a global regulator of biofilm formation, stress adaptation, and virulence in Burkholderia pseudomallei

## TL;DR

The IrlS2-IrlR2 system in Burkholderia pseudomallei regulates biofilm formation, stress adaptation, and virulence, playing a key role in the pathogen's survival and disease-causing potential.

## Contribution

The study identifies and characterizes the previously unstudied IrlS2-IrlR2 two-component system as a global regulator in Burkholderia pseudomallei.

## Key findings

- Knockout of irlR2 leads to enhanced biofilm formation, reduced motility, and increased sensitivity to oxidative stress.
- The ΔirlR2 mutant shows impaired adhesion and cytotoxicity toward A549 cells and attenuated virulence in Galleria mellonella.
- Transcriptomic analysis reveals altered regulation of secretion systems, iron homeostasis, and redox balance in the mutant strain.

## Abstract

Burkholderia pseudomallei, which causes melioidosis, is an adaptable pathogen that survives in diverse environments. Two-component systems (TCSs) play pivotal roles in bacterial signal transduction and adaptation, yet the functions of most TCSs in B. pseudomallei remain poorly characterized. Here, we identified and functionally characterized a previously unstudied TCS, IrlS2-IrlR2, which shares partial homology with the IrlS-IrlR system but exhibits distinct regulatory roles. Knockout of irlR2 (ΔirlR2) did not affect growth in nutrient-rich medium but led to enhanced biofilm formation, reduced motility, decreased siderophore production, and increased sensitivity to oxidative stress, all of which were restored in the complemented strain. The mutant also exhibited growth retardation under subinhibitory cobalt concentrations, despite unchanged MICs. In infection assays, ΔirlR2 displayed impaired adhesion and cytotoxicity toward A549 cells and attenuated virulence in Galleria mellonella, with a higher median lethal dose than the wild type. Transcriptomic analysis revealed hcp2 within the type VI secretion system 2 (T6SS-2) cluster, and multiple T3SS-3 genes were strongly downregulated, consistent with reduced intracellular survival, whereas T3SS-1 and T3SS-2 were upregulated, suggesting a dysregulated secretion system balance. Adhesion-related (fimA) and iron transport (fhuBCDF) genes were repressed, while oxidative stress-associated (cydABX) and nitrate reductase (narIJHGK-nasA) operons were induced, indicating altered regulation of iron homeostasis, redox balance, and nitrogen metabolism, which may reflect adaptive responses to environmental stress. Collectively, these results demonstrate that the IrlS2-IrlR2 system functions as a global regulator, integrating biofilm formation, stress adaptation, and virulence regulation, highlighting its role in the environmental resilience and pathogenic potential of B. pseudomallei.

Burkholderia pseudomallei, which causes melioidosis, poses a serious threat to human and animal health in tropical and subtropical regions worldwide. Classified as a tier 1 biothreat agent by the U.S. CDC and a category II pathogen in China, B. pseudomallei causes severe pneumonia and septicemia with case-fatality rates approaching 50%. Despite its medical and epidemiological significance, the regulatory mechanisms controlling its virulence and environmental persistence remain poorly understood. This study identifies IrlS2-IrlR2 as a previously uncharacterized two-component system (TCS) that acts as a global regulator integrating biofilm formation, stress adaptation, and virulence. Functional and transcriptomic analyses reveal that IrlS2-IrlR2 modulates secretion systems, iron homeostasis, and redox balance. These findings deepen our understanding of B. pseudomallei pathogenesis and highlight the role of TCS-mediated regulatory networks.