Proteomics reveal temperature-coupled cobalamin homeostasis and pathogenicity in Pseudomonas aeruginosa

The opportunistic pathogen Pseudomonas aeruginosa is highly adaptable to different environmental conditions due to its versatile sensing and metabolic capabilities. Both external temperature and metal availability have a strong influence on the virulence and pathogenicity of P. aeruginosa , but the coupling between these two factors is not well understood. While iron is recognized as major player in nutritional immunity, the role of cobalt and the cobalt-containing vitamin B ₁₂ (cobalamin) during host infection remains unclear. Here, we investigate the environmental isolate P. aeruginosa PA254 using high-resolution global proteomics and cellular cobalamin measurements over a temperature gradient spanning environmental, host-associated, and heat-stress conditions (22-42 °C). PA254 occupies a continuum between an ambient-temperature virulent state characterized by versatile secreted factors, exopolysaccharide-rich biofilms, and planktonic swimmers and surface swarmers; and a host-associated virulent state characterized by potent secretion effectors, alginate-dominated biofilms, and a strong proportion of surface twitching motility. Pathway analyses indicate a shift toward carbon sparing, energy conservation, redox control, and metabolic maintenance during a host-adapted lifestyle, along with the strong overexpression of alternative iron acquisition strategies relying on heme and siderophores. Proteins of the cobalamin biosynthetic pathway declined significantly above ambient temperatures, despite constant intracellular B ₁₂ concentrations across all conditions. This decoupling of biosynthesis from cellular pools implies prioritization and recycling within B ₁₂ -dependent processes, while the lack of B ₁₂ production at human body temperatures creates avenues for therapeutics interfering with B ₁₂ supply. Altogether, this work highlights a gradual rather than stepwise reprogramming of the P. aeruginosa proteome in response to environmental cues, and highlights proteomics as a tool to investigate system level mechanisms of challenging pathogens.