Ca2+ regulated antibiotic resistance in Pseudomonas aeruginosa

Pseudomonas aeruginosa, an opportunistic pathogen, causes life threatening infections in cystic fibrosis (CF) patients. Due to increased abundance of Ca2+ in CF lung, P. aeruginosa is surrounded with elevated Ca2+ that could be recognized by the bacterium as a cue for adaptation in this environment. Previous research by our group and others identified Ca2+ responsive regulators and defined their role in Ca2+-dependent virulence factor production. In addition, tightly maintained intracellular Ca2+ ([Ca2+]in) homeostasis suggests the signaling role of Ca2+ in P. aeruginosa. Our current study report that growth at 5 mM/ 10 mM Ca2+ increases the antibiotic resistance of PAO1 more than 10 fold. Here, our main goal was to elucidate the regulatory role of Ca2+ in adaptive antimicrobial resistance and virulence of PAO1. We identified several of the RND superfamily efflux pumps involved in Ca2+-regulated tobramycin resistance, plant infectivity and [Ca2+]in homeostasis maintenance. We have established that Ca2+ transcriptionally regulates five of the six efflux pumps involved in Ca2+-induced tobramycin resistance in a growth phase dependent manner. Ca2+ reliant tobramycin resistance and increase transcription of mexAB-oprM, one of the efflux pumps involved in Ca2+-induced tobramycin resistance, requires intact Ca2+ homeostasis. We have also identified a putative calcium channel in PAO1, homologous to the pH-sensitive Ca2+ leak channel, BsYetJ in Bacillus subtilis. This channel is essential for PAO1 to generate transient changes in [Ca2+]in. Disruption of this gene affects the Ca2+ regulated global transcription of many virulence and adaptation associated genes in PAO1. The lack of intact [Ca2+]in transients also resulted into reduction in Ca2+ regulated virulence of this organism. Previously our lab identified calmodulin-like EF hand protein (EfhP), Ca2+-regulated two-component system (CarSR), Ca2+ binding protein (CarP), and Ca2+-regulated OB-fold protein, which contribute to Ca2+ - regulated virulence in PAO1. Here, we established that Ca2+ regulated transcription of calC is dependent on CarSR, CarP and EfhP. Finally, we also identified three hypothetical proteins involved in Ca2+-induced polymyxin B resistance of PAO1. Overall, the findings of this research identifies the genes involved in Ca2+ regulatory cascade of P. aeruginosa and how they contribute to Ca2+ regulated antibiotic resistance and virulence of this pathogen