Host-derived stimuli alter intracellular calcium levels in Pseudomonas aeruginosa, revealing its potential role as a second messenger

Calcium signaling is a fundamental cellular communication mechanism, well established in eukaryotes, that relies on calcium ions (Ca 2+ ) acting both as first and second messengers to regulate diverse life-sustaining processes. In bacteria, although the significance of Ca 2+ regulation is recognized, it is commonly attributed to its role as a first messenger, while the mechanistic basis for its capacity as a second messenger remains elusive. Previously, we have shown that Pseudomonas aeruginosa, an opportunistic pathogen causing life-threatening infections in humans, possesses the key prerequisites for intracellular Ca 2+ (Ca 2+ in ) signaling, including tightly controlled Ca 2+ in homeostasis, transient changes in Ca 2+ in levels in response to exogenous Ca 2+ , and Ca 2+ -regulated gene expression. In addition, we and others identified several key components of the Ca 2+ signaling network , including Ca 2+ sensors, transporters, and regulators. The remaining question is whether the levels of Ca 2+ in alter in response to any extracellular stimuli other than Ca 2+ . To determine this, we have used a cytoplasmic Ca 2+ probe, R-GECO1, that produces a Ca 2+ -dependent fluorescence and tested a variety of extracellular stimuli, including ions, monosaccharides, amino acids, nucleotides, quorum-sensing signaling molecules, reactive oxygen species, and antibiotics. We observed changes in Ca 2+ in levels in response to several molecules, particularly glucose, asparagine, ATP, ADP, AMP, HOCl, and cycloserine. The results provide the first experimental evidence that, like eukaryotes, P. aeruginosa responds to select, chemically diverse host-related stimuli by altering its Ca 2+ in levels, supporting a potential signaling role of Ca² + in in bacteria. IMPORTANCE Cells use signaling to communicate and respond to their environment. Although the role of calcium ions (Ca² + ) as a signaling molecule, regulating multiple essential processes, has been well established in eukaryotes, Ca² + signaling in bacteria remains understudied. Previous research has indicated the role of Ca² + in bacterial regulation as a first messenger; however, its role as a second messenger is not yet recognized. Here, we show, for the first time, that P. aeruginosa , an important human pathogen, alters its intracellular Ca² + levels in response to various external stimuli commonly associated with the host environment. These findings provide the mechanistic basis for P. aeruginosa to use Ca² + as a second messenger. Understanding these Ca 2+ responses provides novel insights into Ca 2+ -mediated adaptation of invading pathogens to the host environment and may inform the development of new antimicrobial strategies.