Calcium induces tobramycin resistance in Pseudomonas aeruginosa by regulating RND efflux pumps.

Pseudomonas aeruginosa is an opportunistic multidrug resistant pathogen causing severe chronic infections. Our previous studies showed that elevated calcium (Ca) enhances production of several virulence factors and plant infectivity of the pathogen. Here we show that Ca increases resistance of P. aeruginosa PAO1 to tobramycin, antibiotic commonly used to treat Pseudomonas infections. LC-MS/MS-based comparative analysis of the membrane proteomes of P aeruginosa grown at elevated versus not added Ca, determined that the abundances of two RND (resistance-nodulation-cell division) efflux pumps, MexAB-OprM and MexVW-OprM, were increased in the presence of elevated Ca. Analysis of twelve transposon mutants with disrupted RND efflux pumps showed that six of them (mexB, muxC, mexY, mexJ, czcB, and mexE) contribute to Ca-induced tobramycin resistance. Transcriptional analyses by promoter activity and RT-qPCR showed that the expression of mexAB, muxABC, mexXY, mexJK, czcCBA, and mexVW is increased by elevated Ca. Disruption of mexJ, mexC, mexI, and triA significantly decreased Ca-induced plant infectivity of the pathogen. Earlier, our group showed that PAO1 maintains intracellular Ca (Ca) homeostasis, which mediates Ca regulation of P. aeruginosa virulence, and identified four putative Ca transporters involved in this process (Guragain et al., 2013). Here we show that three of these transporters (PA2435, PA2092, PA4614) play role in Ca-induced tobramycin resistance and one of them (PA2435) contributes to Ca regulation of mexAB-oprM promoter activity. Furthermore, mexJ, czcB, and mexE contribute to the maintenance of Ca homeostasis. This provides the first evidence that Ca homeostasis mediates Ca regulation of RND transport systems, which contribute to Ca-enhanced tobramycin resistance and plant infectivity in P. aeruginosa.