# Deletion of tonB1 in Pseudomonas aeruginosa impairs zinc homeostasis and pathogenicity

## TL;DR

Deleting the TonB1 gene in Pseudomonas aeruginosa disrupts zinc balance and reduces its ability to cause disease.

## Contribution

TonB1 is shown to be essential for zinc homeostasis and virulence in Pseudomonas aeruginosa.

## Key findings

- Deleting tonB1 reduces zinc uptake and intracellular zinc levels in Pseudomonas aeruginosa.
- The tonB1 mutant shows increased susceptibility to calprotectin and reduced virulence in infection models.
- TonB1 is critical for bacterial fitness in Galleria mellonella hemolymph under zinc limitation.

## Abstract

Many bacterial pathogens must acquire metal ions for proliferation and pathogenesis. In gram-negative bacteria, the TonB system is crucial for nutrient uptake. Previous research indicates that Pseudomonas aeruginosa uses the energy transduction protein TonB1 for iron uptake. Although zinc and iron are essential for P. aeruginosa, it is unknown whether TonB1 is also important for its zinc uptake. Here, a tonB1 deletion mutant was constructed from P. aeruginosa PAO1. Inductively coupled plasma mass spectrometry and other methods revealed that the tonB1 mutation significantly altered zinc homeostasis, as evidenced by diminished zinc uptake capacity, and affected other zinc-related phenotypes in P. aeruginosa, such as increased susceptibility to the host-secreted nutritional immunity protein calprotectin (CP), reduced oxidative stress resistance, impaired motility, and attenuated virulence in both Chinese cabbage and Galleria mellonella larvae, and resulted in reduced bacterial fitness in G. mellonella hemolymph. These findings underscore the critical role of tonB1 in zinc homeostasis and associated phenotypes in P. aeruginosa.

Zinc is the second most abundant metal element in cells, and it plays an important role in the pathogenicity and antibiotic resistance of pathogenic bacteria. Pseudomonas aeruginosa is an increasingly prevalent and multidrug-resistant pathogen that relies on TonB proteins for transporting numerous nutrients. Herein, we revealed that TonB1 is essential for zinc homeostasis in P. aeruginosa; its deletion severely impaired bacterial growth under zinc limitation and was associated with reduced intracellular zinc levels and dysregulation of zinc uptake-related genes—potentially contributing to heightened susceptibility to host defenses (e.g., calprotectin), oxidative stress, and loss of motility and infectivity. This discovery highlights a critical role for TonB1 in maintaining zinc homeostasis, which impacts pathogenicity in P. aeruginosa. Although TonB homologs have been implicated in zinc uptake elsewhere, our work demonstrates that it is indispensable for virulence in this pathogen, significantly expanding the understanding of TonB's physiological functions beyond iron uptake and highlighting a key adaptation mechanism for essential metal nutrients.