An HWE-Family Histidine Kinase Modulates Brucella Cell Envelope Properties and Host Innate Immune Response

The bacterial cell envelope is essential for viability and host interaction. In the intracellular pathogen Brucella ovis, the orphan HWE-family histidine kinase PhyK has been implicated in processes that influence cell envelope homeostasis, yet its function remains largely uncharacterized. We show that deletion of phyK (∆phyK) disrupts cell size control, increases resistance to anionic detergents, enhances sensitivity to cationic envelope disruptors, and triggers broad transcriptional changes, including reduced expression of aerobic respiration genes and increased expression of genes involved in transport and lipid metabolism. This transcriptional profile mirrors that of wild-type B. ovis exposed to an anionic detergent, indicating that loss of PhyK function primes cells to resist this stress. Despite its altered cell envelope properties, the ∆phyK mutant exhibits no fitness defect in ex vivo macrophage infection models. However, it elicits a significantly reduced pro-inflammatory cytokine response in activated murine macrophages compared to the wild-type strain. We further show that purified PhyK can form multiple stable oligomeric species in solution, reflecting the structural plasticity observed in other HWE-family kinases and likely contributing to its signaling function in vivo. Our results establish PhyK as a key regulator of B. ovis cell envelope properties that can modulate host immune interactions.